This invention relates to EP4 receptor selective prostaglandin agonists, combinations, methods, kits and pharmaceutical compositions comprising said prostaglandin agonists which are useful to prevent bone loss, restore or augment bone mass and to enhance bone healing including the treatment of conditions which present with low bone mass and/or bone defects in vertebrates, and particularly mammals, including humans.
Osteoporosis is a systemic skeletal disease, characterized by low bone mass and deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. In the U.S., the condition affects more than 25 million people and causes more than 1.3 million fractures each year, including 500,000 spine, 250,000 hip and 240,000 wrist fractures annually. Hip fractures are the most serious consequence of osteoporosis, with 5-20% of patients dying within one year, and over 50% of survivors being incapacitated.
The elderly are at greatest risk of osteoporosis, and the problem is therefore predicted to increase significantly with the aging of the population. Worldwide fracture incidence is forecasted to increase three-fold over the next 60 years, and one study has estimated that there will be 4.5 million hip fractures worldwide in 2050.
Women are at greater risk of osteoporosis than men. Women experience a sharp acceleration of bone loss during the five years following menopause. Other factors that increase the risk include smoking, alcohol abuse, a sedentary lifestyle and low calcium intake.
There are currently two main types of pharmaceutical therapy for the treatment of osteoporosis. The first is the use of anti-resorptive compounds to reduce the resorption of bone tissue.
Estrogen is an example of an anti-resorptive agent. It is known that estrogen reduces fractures. In addition, Black, et al. in EP 0605193A1 report that estrogen, particularly when taken orally, lowers plasma levels of LDL and raises those of the beneficial high density lipoproteins (HDL""s). However, estrogen fails to restore bone back to young adult levels in the established osteoporotic skeleton. Furthermore, long-term estrogen therapy has been implicated in a variety of disorders, including an increase in the risk of uterine cancer, endometrial cancer and possibly breast cancer, causing many women to avoid this treatment. The significant undesirable effects associated with estrogen therapy support the need to develop alternative therapies for osteoporosis that have the desirable effect on serum LDL but do not cause undesirable effects.
A second type of pharmaceutical therapy for the treatment of osteoporosis is the use of anabolic agents to promote bone formation and increase bone mass. This class of agents is expected to restore bone to the established osteoporotic skeleton.
In addition to osteoporosis, approximately, 20-25 million women and an increasing number of men have detectable vertebral fractures as a consequence of reduced bone mass, with an additional 250,000 hip fractures reported yearly in America alone. The latter case is associated with a 12% mortality rate within the first two years and with a 30% rate of patients requiring nursing home care after the fracture. While this is already significant, the economic and medical consequences of convalescence due to slow or imperfect healing of these bone fractures is expected to increase, due to the aging of the general population.
Estrogens have been shown (Bolander et al., 38th Annual Meeting Orthopedic Research Society, 1992) to improve the quality of the healing of appendicular fractures. Therefore, estrogen replacement therapy should be effective as a method for the treatment of fracture repair. However, patient compliance with estrogen therapy is relatively poor due to its side effects, including the resumption of menses, mastodynia, an increased risk of uterine cancer, an increased perceived risk of breast cancer, and the concomitant use of progestins. In addition, men are likely to object to the use of estrogen treatment. The need exists for a therapy which would be beneficial to patients who have suffered debilitating bone fractures and which would increase patient compliance.
It has been demonstrated that prostaglandin E2 (PGE2) can restore lost bone in an ovariectomized (OVX) rat model, a model for postmenopausal osteoporosis. Ke, H. Z., et al., Bone, 23:249-255, 1998. However there are severe side effects associated with PGE2. Jee, W. S. S. and Ma, Y. F., Bone, 21:297-304, 1997.
Great Britain Patent Specification 1 553 595 discloses compounds of the formula 
wherein the double bonds are cis or trans and the variables are defined as set forth therein. Those compounds are disclosed as having spasmogenic and spasmolytic activity, for example bronchodilatory and antihypertensive effects. The compounds are also disclosed as having utility in the inhibition of the secretion of gastric juice and as having abortive effects.
U.S. Pat. No. 4,115,401 discloses compound of the formula 
wherein the variables are defined as set forth therein. Those compounds are disclosed as having spasmogenic, cardiovascular and bronchodilatory effects.
U.S. Pat. No. 4,113,873 discloses compound of the formula 
wherein the variables are defined as set forth therein. Those compounds are disclosed as having utility as a bronchodilator, as an antihypertensive agent, as an enhancer of spontaneous contraction of the uterus and for the treatment of gastro-intestinal disorders or gastric ulcers.
Great Britain Patent Specification 1 583 163 discloses compounds of the formula 
wherein the variables are defined as set forth therein. Those compounds are disclosed as having spasmogenic, bronchodilatory, vasoconstricting, vasodilating and abortive properties as well as utility in the inhibition of gastric acid secretion.
Commonly assigned U.S. Pat. No. 4,177,346 discloses compounds of the formula 
wherein the variables are defined as set forth therein. Those compounds are disclosed as having vasodilator, antihypertensive, bronchodilator, antifertility and antisecretory activity.
International Patent Application Publication No. WO00 21542 discloses that EP4 receptor subtype agonists have utility as stimulators of bone formation.
Although there are a variety of osteoporosis therapies, there is a continuing need and a continuing search in this field of art for alternative osteoporosis therapies. In addition, there is a need for bone fracture healing therapies. Also, there is a need for therapy which can promote bone re-growth into skeletal areas where defects exist such as defects caused or produced by, for example, tumors in bone. Further, there is a need for therapy which can promote bone re-growth into skeletal areas where bone graft surgery has been completed.
This invention is directed to compounds of Formula I 
prodrugs thereof, pharmaceutically acceptable salts of said compounds and said prodrugs and stereoisomers and diastereomeric mixtures of said compounds, prodrugs and salts, wherein the dotted line is a bond or no bond; X is xe2x80x94CH2xe2x80x94 or O; Z is xe2x80x94(CH2)3xe2x80x94, thienyl, thiazolyl or phenyl, provided that when X is O, then Z is phenyl; Q is carboxyl, (C1-C4)alkoxylcarbonyl or tetrazolyl; R2 is xe2x80x94Ar or xe2x80x94Ar1xe2x80x94Vxe2x80x94Ar2; V is a bond, xe2x80x94Oxe2x80x94, xe2x80x94OCH2xe2x80x94 or
xe2x80x94CH2Oxe2x80x94;
Ar is a partially saturated, fully saturated or fully unsaturated five to eight membered ring optionally having one to four heteroatoms selected independently from oxygen, sulfur and nitrogen, or a bicyclic ring consisting of two fused independently partially saturated, fully saturated or fully unsaturated five or six membered rings, taken independently, optionally having one to four heteroatoms selected independently from nitrogen, sulfur and oxygen, said partially or fully saturated ring or bicyclic ring optionally having one or two oxo groups substituted on carbon or one or two oxo groups substituted on sulfur; and
Ar1 and Ar2 are each independently a partially saturated, fully saturated or fully unsaturated five to eight membered ring optionally having one to four heteroatoms selected independently from oxygen, sulfur and nitrogen, said partially or fully saturated ring optionally having one or two oxo groups substituted on carbon or one or two oxo groups substituted on sulfur;
said Ar moiety is optionally substituted on carbon or nitrogen, on one ring if the moiety is monocyclic, or on one or both rings if the moiety is bicyclic, with up to three substituents per ring each independently selected from hydroxy, halo, carboxy, (C1-C7)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, (C1-C7)alkyl, (C2-C7)alkenyl, (C3-C7)cycloalkyl, (C3-C7)cycloalkyl(C1-C4)alkyl, (C3-C7)cycloalkyl(C1-C4)alkanoyl, formyl, (C1-C8)alkanoyl, (C1-C6)alkanoyl(C1-C6)alkyl, (C1-C4)alkanoylamino, (C1-C4)alkoxycarbonylamino, hydroxysulfonyl, aminocarbonylamino or mono-Nxe2x80x94, di-N,N-, di-N,Nxe2x80x2- or tri-N,N,Nxe2x80x2-(C1-C4)alkyl substituted aminocarbonylamino, sulfonamido, (C1-C4)alkylsulfonamido, amino, mono-N- or di-N,N-(C1-C4)alkylamino, carbamoyl, mono-N- or di-N,N-(C1-C4)alkylcarbamoyl, cyano, thiol, (C1-C6)alkylthio, (C1-C6)alkylsulfinyl, (C1-C4)alkylsulfonyl and mono-N- or di-N,N-(C1-C4)alkylaminosulfinyl, wherein said alkyl and alkoxy substituents in the definition of Ar are optionally substituted on carbon with up to three fluoro;
said Ar1 and Ar2 moieties are independently optionally substituted on carbon or nitrogen with up to three substituents each independently selected from hydroxy, halo, carboxy, (C1-C7)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, (C1-C7)alkyl, (C2-C7)alkenyl, (C3-C7)cycloalkyl, (C3-C7)cycloalkyl(C1-C4)alkyl, (C3-C7)cycloalkyl(C1-C4)alkanoyl, formyl, (C1-C8)alkanoyl, (C1-C6)alkanoyl(C1-C6)alkyl, (C1-C4)alkanoylamino, (C1-C4)alkoxycarbonylamino, hydroxysulfonyl, aminocarbonylamino or mono-N-, di-N,N-, di-N,Nxe2x80x2- or tri-N,N,Nxe2x80x2-(C1-C4)alkyl substituted aminocarbonylamino, sulfonamido, (C1-C4)alkylsulfonamido, amino, mono-N- or di-N,N-(C1-C4)alkylamino, carbamoyl, mono-N- or di-N,N-(C1-C4)alkylcarbamoyl, cyano, thiol, (C1-C6)alkylthio, (C1-C6)alkylsulfinyl, (C1-C4)alkylsulfonyl and mono-N- or di-N,N-(C1-C4)alkylaminosulfinyl, wherein said alkyl and alkoxy substituents in the definition of Ar1 and Ar2 are optionally substituted on carbon with up to three fluoro;
provided that (a) when X is (CH2)xe2x80x94 and Z is xe2x80x94(CH2)3xe2x80x94, then R2 is not thienyl, phenyl or phenyl monosubstituted with chloro, fluoro, phenyl, methoxy, trifluoromethyl or (C1-C4)alkyl; and (b) when X is (CH2)xe2x80x94, Z is xe2x80x94(CH2)3xe2x80x94, and Q is carboxyl or (C1-C4)alkoxycarbonyl, then R2 is not (i) (C5-C7)cycloalkyl or (ii) phenyl, thienyl or furyl each of which may be optionally monosubstituted or disubstituted by one or two substituents selected, independently in the latter case, from halogen atoms, alkyl groups having 1-3 carbon atoms which may be substituted by one or more halogen atoms, and alkoxy groups having 1-4 carbon atoms.
A preferred group of compounds, designated Group A, are those compounds of the Formula Ia, 
prodrugs thereof, pharmaceutically acceptable salts of said compounds and said prodrugs, and stereoisomers and diastereomeric mixtures of said compounds, prodrugs and salts, wherein: X is xe2x80x94CH2xe2x80x94; Z is xe2x80x94(CH2)3xe2x80x94, 
and R2 is Ar wherein said Ar moiety is optionally substituted on carbon or nitrogen, on one ring if the moiety is monocyclic, or on one or both rings if the moiety is bicyclic, with up to three substituents per ring each independently selected from hydroxy, halo, carboxy, (C1-C7)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, (C1-C7)alkyl, (C2-C7)alkenyl, (C3-C7)cycloalkyl, (C3-C7)cycloalkyl(C1-C4)alkyl, (C3-C7)cycloalkyl(C1-C4)alkanoyl, formyl, (C1-C8)alkanoyl, (C1-C6)alkanoyl(C1-C6)alkyl, (C1-C4)alkanoylamino, (C1-C4)alkoxycarbonylamino, hydroxysulfonyl, aminocarbonylamino or mono-N-, di-N,N-, di-N,Nxe2x80x2- or tri-N,N,Nxe2x80x2-(C1-C4)alkyl substituted aminocarbonylamino, sulfonamido, (C1-C4)alkylsulfonamido, amino, mono-N- or di-N,N-(C1-C4)alkylamino, carbamoyl, mono-N- or di-N,N-(C1-C4)alkylcarbamoyl, cyano, thiol, (C1-C6)alkylthio, (C1-C6)alkylsulfinyl, (C1-C4)alkylsulfonyl and mono-N- or di-N,N-(C1-C4)alkylaminosulfinyl, wherein said alkyl and alkoxy substituents in the definition of Ar are optionally substituted on carbon with up to three fluoro.
A preferred group of compounds within Group A, designated Group B, are those compounds, prodrugs thereof, pharmaceutically acceptable salts of said compounds and said prodrugs, and stereoisomers and diastereomeric mixtures of said compounds, prodrugs and salts wherein Ar is cyclohexyl, 1,3-benzodioxolyl, thienyl, naphthyl or phenyl optionally substituted with one or two (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, chloro, fluoro, trifluoromethyl or cyano, wherein said alkyl and alkoxy substituents in the definition of Ar are optionally substituted with up to three fluoro.
A preferred group of compounds within Group B, designated Group C, are those compounds, prodrugs thereof, pharmaceutically acceptable salts of said compounds and said prodrugs, and stereoisomers and diastereomeric mixtures of said compounds, prodrugs and salts wherein the dotted line is no bond; Q is carboxy or (C1-C4)alkoxylcarbonyl; and Z is 
A preferred group of compounds within Group C, designated Group D, are those compounds, prodrugs thereof and pharmaceutically acceptable salts of said compounds and said prodrugs, and stereoisomers and diastereomeric mixtures of said compounds, prodrugs and salts wherein Q is carboxy and Ar is phenyl optionally substituted with one (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, chloro, fluoro, trifluoromethyl or cyano, wherein said alkyl and alkoxy substituents in the definition of Ar are optionally substituted with up to three fluoro.
A preferred compound within Group D is the compound, prodrugs thereof and pharmaceutically acceptable salts of said compound and said prodrugs, and stereoisomers and diastereomeric mixtures of said compound, prodrugs and salts wherein Ar is m-trifluoromethylphenyl.
Another preferred compound within Group D is the compound, prodrugs thereof and pharmaceutically acceptable salts of said compound and said prodrugs, and stereoisomers and diastereomeric mixtures of said compound, prodrugs and salts wherein Ar is m-chlorophenyl.
Another preferred compound within Group D is the compound, prodrugs thereof and pharmaceutically acceptable salts of said compound and said prodrugs, and stereoisomers and diastereomeric mixtures of said compound, prodrugs and salts wherein Ar is m-trifluoromethoxyphenyl.
An especially preferred group of compounds of this invention include 5-(3-(2S-(3R-hydroxy-4-(3-trifluoromethyl-phenyl)-butyl)-5-oxo-pyrrolidin-1-yl)-propyl)-thiophene-2-carboxylic acid; 5-(3-(2S-(3R-hydroxy-4-(3-trifluoromethoxy-phenyl)-butyl)-5-oxo-pyrrolidin-1-yl)-propyl)-thiophene-2-carboxylic acid; and 5-(3-(2S-(4-(3-chloro-phenyl)-3R-hydroxy-butyl)-5-oxo-pyrrolidin-1-yl)-propyl)-thiophene-2-carboxylic acid.
Another preferred group of compounds within Group A are those compounds of Group A, pharmaceutically acceptable salts of said compounds and said prodrugs and stereoisomers and diatereomeric mixtures of said compounds, prodrugs and salts, wherein X is xe2x80x94CH2xe2x80x94, Z is xe2x80x94(CH2)3xe2x80x94, Q is carboxyl or (C1-C4)alkoxycarbonyl and Ar is phenyl independently substituted with one to three cyano, (C1-C7)alkoxy substituted with one to three fluoro or (C1-C4)alkoxy(C1-C4)alkyl.
This invention is particularly directed to a compound of Formula I as defined in the immediately preceeding paragraph, pharmaceutically acceptable salts of said compounds and said prodrugs and stereoisomers and diatereomeric mixtures of said compounds, prodrugs and salts, wherein the dotted line is no bond; Q is carboxy or (C1-C4)alkoxylcarbonyl; and Z is 
This invention is particularly directed to a compound of Formula I as defined in the immediately preceeding paragraph, a prodrug thereof, pharmaceutically acceptable salts of said compounds and said prodrugs and stereoisomers and diatereomeric mixtures of said compounds, prodrugs and salts, wherein Q is carboxy and Ar is phenyl optionally substituted with one (C1-C4)alkyl, (C1-C4)alkoxy, (C1-C4)alkoxy(C1-C4)alkyl, chloro, fluoro, trifluoromethyl or cyano, wherein said alkyl and alkoxy substituents in the definition of Ar are optionally substituted with up to three fluoro.
This invention is further directed to methods of treating a condition which presents with low bone mass in a mammal comprising administering to said mammal an EP4 receptor selective compound of Formula I, a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug, or a stereoisomer or diastereomeric mixture of said compound, salt or prodrug.
This invention is particularly directed to such methods wherein said condition is osteoporosis, frailty, an osteoporotic fracture, a bone defect, childhood idiopathic bone loss, alveolar bone loss, mandibular bone loss, bone fracture, osteotomy, bone loss associated with periodontitis, or prosthetic ingrowth. In preferred methods of this invention, the EP4 receptor selective agonist is administered systemically. In other preferred methods of this invention, the EP4 agonist is administered locally.
This invention is particularly directed to such methods wherein such condition is a metastable bone disease wherein surgical removal of bone leaves a bone defect which requires filling.
The methods of this invention are especially useful wherein said condition is frailty.
The methods of this invention are also especially useful wherein said condition is osteoporosis.
The methods of this invention are also especially useful wherein said condition is bone fracture or osteoporotic fracture.
This invention is also directed to pharmaceutical compositions comprising a compound of Formula I, a prodrug thereof, a pharmaceutically acceptable salt of said compound or said prodrug, or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt of this invention and a pharmaceutically acceptable carrier, vehicle or diluent. This invention is also directed to methods of treating a condition which presents with low bone mass in a mammal comprising administering to said mammal such a pharmaceutical composition.
Preferably post-menopausal women and men over the age of 60 are treated. Also preferred are individuals regardless of age who have significantly reduced bone mass, i.e., greater than or equal to 1.5 standard deviations below young normal levels.
In the methods of this invention, conditions which present with low bone mass include such conditions as, for example, osteoporosis, childhood idiopathic bone loss, alveolar bone loss, mandibular bone loss, bone fracture, osteotomy, bone loss associated with periodontitis and prosthetic ingrowth.
Methods for treating xe2x80x9csecondary osteoporosisxe2x80x9d are also included within the methods of this invention. xe2x80x9cSecondary osteoporosisxe2x80x9d includes glucocorticoid-induced osteoporosis, hyperthyroidism-induced osteoporosis, immobilization-induced osteoporosis, heparin-induced osteoporosis and immunosuppressive-induced osteoporosis in a vertebrate, e.g., a mammal (including a human being). These methods are carried out by administering to said vertebrate, e.g., mammal, a xe2x80x9csecondary osteoporosisxe2x80x9d treating amount of an EP4 receptor selective prostaglandin agonist of Formula I, a prodrug thereof or a pharmaceutically acceptable salt of said EP4 receptor selective prostaglandin agonist or of said prodrug or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt.
Yet another aspect of this invention is directed to methods for strengthening a bone graft, inducing vertebral synostosis, enhancing long bone extension, enhancing bone healing following facial reconstruction, maxillary reconstruction and/or mandibular reconstruction in a vertebrate, e.g., a mammal (including a human being), comprising administering to said vertebrate, e.g., a mammal which has undergone bone graft surgery, induction of vertebral synostosis, enhancement of long bone extension, facial reconstruction, maxillary reconstruction or mandibular reconstruction, a bone enhancing amount of an EP4 receptor selective prostaglandin agonist of Formula I, a prodrug thereof or a pharmaceutically acceptable salt of said EP4 receptor selective prostaglandin agonist or of said prodrug, or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt. The EP4 receptor selective prostaglandin agonists of this invention may be applied locally to the site of bone reconstruction or may be administered systemically.
This invention is also directed to a method of treating impotence or erectile dysfunction which comprises administering to a patient in need of such treatment an impotence or erectile dysfunction treating amount of a compound of Formula I, a prodrug thereof, a pharmaceutically acceptable salt of said compound or said prodrug or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt.
This invention is also directed to a method for treating a mammal which presents with impaired renal function comprising administering to said mammal a kidney regenerating effective amount of a compound of Formula I, a prodrug thereof, a pharmaceutically acceptable salt of said compound or said prodrug or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt.
This invention is also directed to methods of promoting bone growth comprising administering to a mammal a therapeutically effective amount of a compound of Formula I, a prodrug thereof, a pharmaceutically acceptable salt of said compound or said prodrug or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt; and a therapeutically effective amount of a HMG-CoA reductase inhibitor (statin) or a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug.
A preferred dosage is about 0.001 to about 100 mg/kg/day of a compound of Formula I, a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug, or a stereoisomer or a diastereomeric mixture of said compound, prodrug or salt. An especially preferred dosage is about 0.01 to about 10 mg/kg/day of a compound of Formula I, a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug, or a stereoisomer or a diastereomeric mixture of said compound, prodrug or salt.
Yet another aspect of this invention is directed to combinations of a Formula I compound, a prodrug thereof or a pharmaceutically acceptable salt of said compound or of said prodrug, or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt, and other compounds as described below.
Yet another aspect of this invention is directed to pharmaceutical compositions comprising a compound of Formula I, a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug, or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt, and an anti-resorptive agent, a prodrug thereof or a pharmaceutically acceptable salt of said agent or said prodrug and for the use of such compositions for the treatment or prevention of conditions which present with low bone mass, including osteoporosis in a vertebrates, e.g., mammals (e.g., humans, particularly women) or the use of such compositions for other bone mass augmenting uses.
The combinations of this invention comprise a therapeutically effective amount of a first compound, said first compound being a Formula I compound, a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug, or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt; and a therapeutically effective amount of a second compound, said second compound being an anti-resorptive agent, a prodrug thereof or a pharmaceutically acceptable salt of said agent or said prodrug such as an estrogen agonist/antagonist or a bisphosphonate.
Another aspect of this invention is directed to methods for treating vertebrates, e.g., mammals which present with low bone mass comprising administering to said vertebrate, e.g., a mammal having a condition which presents with low bone mass
a. an amount of a first compound, said first compound being a Formula I compound, a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug, or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt; and
b. an amount of a second compound, said second compound being an anti-resorptive agent, a prodrug thereof or a pharmaceutically acceptable salt of said agent or said prodrug such as an estrogen agonist/antagonist or a bisphosphonate.
Such compositions and methods may also be used for other bone mass augmenting uses.
A preferred aspect of this method is wherein the condition which presents with low bone mass is osteoporosis.
Another preferred aspect of this method is wherein the first compound and the second compound are administered substantially simultaneously.
Another aspect of this invention is a kit comprising:
a. an amount of a Formula I compound, a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug, or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt and a pharmaceutically acceptable carrier or diluent in a first unit dosage form;
b. an amount of an anti-resorptive agent, a prodrug thereof or a pharmaceutically acceptable salt of said agent or said prodrug such as an estrogen agonist/antagonist or a bisphosphonate and a pharmaceutically acceptable carrier or diluent in a second unit dosage form; and
c. a container.
Yet another aspect of this invention is directed to pharmaceutical compositions comprising a compound of Formula I, a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug, or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt, and another bone anabolic agent (although the other bone anabolic agent may be a different Formula I compound), a prodrug thereof or a pharmaceutically acceptable salt of said agent or said prodrug and for the use of such compositions for the treatment of conditions which present with low bone mass, including osteoporosis in a vertebrates, e.g., mammals (e.g., humans, particularly women), or the use of such compositions for other bone mass augmenting uses. Such compositions comprise a therapeutically effective amount of a first compound, said first compound being a Formula I compound, a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt; and a therapeutically effective amount of a second compound, said second compound being another bone anabolic agent, a prodrug thereof or a pharmaceutically acceptable salt of said agent or said prodrug.
Another aspect of this invention is directed to methods for treating vertebrates, e.g., mammals which present with low bone mass comprising administering to said vertebrate, e.g., a mammal having a condition which presents with low bone mass
a. an amount of a first compound, said first compound being a Formula I compound, a prodrug thereof or a pharmaceutically acceptable salt or prodrug thereof, or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt; and
b. an amount of a second compound, said second compound being another bone anabolic agent, a prodrug thereof or a pharmaceutically acceptable salt of said agent or said prodrug.
Such compositions and methods may also be used for other bone mass augmenting uses.
A preferred aspect of this method is wherein the condition which presents with low bone mass is osteoporosis.
Another aspect of this invention is a kit comprising:
a. an amount of a Formula I compound, a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug, or a stereoisomer or diastereomeric mixture of said compound, prodrug or salt, and a pharmaceutically acceptable carrier or diluent in a first unit dosage form;
b. an amount of a second compound, said second compound being another bone anabolic agent, a prodrug thereof or a pharmaceutically acceptable salt of said agent or said prodrug in a second unit dosage form; and
c. a container.
Where used in any of the above methods, kits and compositions, certain bone anabolic agents, estrogen agonists/antagonists and bisphosphonates are preferred or especially preferred.
Preferred bone anabolic agents include IGF-1, prostaglandins, prostaglandin agonists/antagonists, sodium fluoride, parathyroid hormone (PTH), active fragments of parathyroid hormone, parathyroid hormone related peptides and active fragments and analogues of parathyroid hormone related peptides, growth hormones or growth hormone secretagogues and the pharmaceutically acceptable salts thereof.
Preferred estrogen agonists/antagonists include droloxifene, raloxifene, tamoxifen; 4-hydroxy-tamoxifen; toremifene; centchroman; levormeloxifene; idoxifene; 6-(4-hydroxy-phenyl)-5-(4-(2-piperidin-1-yl-ethoxy)-benzyl)-naphthalen-2-ol; (4-(2-(2-aza-bicyclo[2.2.1]hept-2-yl)-ethoxy)-phenyl)-(6-hydroxy-2-(4-hydroxy-phenyl)-benzo[b]thiophen-3-yl)-methanone;
3-(4-(1,2-diphenyl-but-1-enyl)-phenyl)-acrylic acid;
2-(4-methoxy-phenyl)-3-[4-(2-piperidin-1-yl-ethoxy)-phenoxy]-benzo[b]thiophen-6-ol;
cis-6-(4-fluoro-phenyl)-5-(4-(2-piperidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol;
(xe2x88x92)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol (lasofoxifene);
cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol;
cis-1-(6xe2x80x2-pyrrolodinoethoxy-3xe2x80x2-pyridyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydronaphthalene;
1-(4xe2x80x2-pyrrolidinoethoxyphenyl)-2-(4xe2x80x3-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;
cis-6-(4-hydroxyphenyl)-5-(4-(2-piperidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol; and
1-(4xe2x80x2-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinoline and the pharmaceutically acceptable salts thereof.
Especially preferred estrogen agonists/antagonists include:
3-(4-(1,2-diphenyl-but-1-enyl)-phenyl)-acrylic acid;
2-(4-methoxy-phenyl)-3-[4-(2-piperidin-1-yl-ethoxy)-phenoxy]-benzo[b]thiophen-6-ol;
cis-6-(4-fluoro-phenyl)-5-(4-(2-piperidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol;
(xe2x88x92)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol (lasofoxifene);
cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol;
cis-1-(6xe2x80x2-pyrrolodinoethoxy-3xe2x80x2-pyridyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydronaphthalene;
1-(4xe2x80x2-pyrrolidinoethoxyphenyl)-2-(4xe2x80x3-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;
cis-6-(4-hydroxyphenyl)-5-(4-(2-piperidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol;
1-(4xe2x80x2-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinoline; and the pharmaceutically acceptable salts thereof.
Preferred bisphosphonates include, tiludronic acid, alendronic acid, zoledronic acid, ibandronic acid, risedronic acid, etidronic acid, clodronic acid, and pamidronic acid and their pharmaceutically acceptable salts.
It will be recognized that prodrugs and pharmaceutically acceptable salts may be formed from the compounds used as the second compounds in the combinations of this invention. All of such prodrugs and pharmaceutically acceptable salts so formed are within the scope of this invention. Particularly preferred salt forms include raloxifene hydrochloride, tamoxifen citrate and toremifene citrate.
The phrase xe2x80x9ccondition(s) which presents with low bone massxe2x80x9d refers to a condition where the level of bone mass is below the age specific normal as defined in standards by the World Health Organization xe2x80x9cAssessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis (1994). Report of a World Health Organization Study Group. World Health Organization Technical Series 843xe2x80x9d. Included in xe2x80x9ccondition(s) which presents with low bone massxe2x80x9d are primary and secondary osteoporosis, as described above. Also included is periodontal disease, alveolar bone loss, post-osteotomy and childhood idiopathic bone loss. The phrase xe2x80x9ccondition(s) which presents with low bone massxe2x80x9d also includes long term complications of osteoporosis such as curvature of the spine, loss of height and prosthetic surgery.
The phrase xe2x80x9ccondition(s) which presents with low bone massxe2x80x9d also refers to a vertebrate, e.g., a mammal, known to have a significantly higher than average chance of developing such diseases as are described above including osteoporosis (e.g., post-menopausal women, men over the age of 50). Other bone mass augmenting or enhancing uses include bone restoration, increasing the bone fracture healing rate, replacing bone graft surgery entirely, enhancing the rate of successful bone grafts, bone healing following facial reconstruction or maxillary reconstruction, mandibular reconstruction, long bone reconstruction, prosthetic ingrowth, vertebral synostosis or long bone extension.
The methods of this invention may also be used in conjunction with orthopedic devices such as spinal fusion cages, spinal fusion hardware, internal and external bone fixation devices, screws and pins.
Those skilled in the art will recognize that the term bone mass actually refers to bone mass per unit area which is sometimes (although not strictly correctly) referred to as bone mineral density.
The term xe2x80x9ctreatingxe2x80x9d, xe2x80x9ctreatxe2x80x9d or xe2x80x9ctreatmentxe2x80x9d as used herein includes preventative (e.g., prophylactic), palliative and curative treatment.
By xe2x80x9cpharmaceutically acceptablexe2x80x9d it is meant the carrier, vehicle, diluent, excipients, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
The expression xe2x80x9cprodrugxe2x80x9d refers to compounds that are drug precursors which, following administration, release the drug in vivo via some chemical or physiological process (e.g., a prodrug on being brought to the physiological pH or through enzyme action is converted to the desired drug form). Exemplary prodrugs upon cleavage release the corresponding drug compound.
The expression xe2x80x9cpharmaceutically acceptable saltxe2x80x9d refers to nontoxic anionic salts containing anions such as, but not limited to, chloride, bromide, iodide, sulfate, bisulfate, phosphate, acetate, maleate, fumarate, oxalate, lactate, tartrate, citrate, gluconate, methanesulfonate and 4-toluene-sulfonate. The expression also refers to nontoxic cationic salts such as, but not limited to, sodium, potassium, calcium, magnesium, ammonium or protonated benzathine (N,Nxe2x80x2-dibenzylethylenediamine), choline, ethanolamine, diethanolamine, ethylenediamine, meglamine (N-methyl-glucamine), benethamine (N-benzylphenethylamine), piperazine or tromethamine (2-amino-2-hydroxymethyl-1,3-propanediol).
The chemist of ordinary skill in the art will also recognize that certain compounds of formula I of this invention can exist in tautomeric form, i.e., that an equilibrium exists between two isomers which are in rapid equilibrium with each other. A common example of tautomerism is keto-enol tautomerism, i.e., 
Examples of compounds which can exist as tautomers include hydroxypyridines, hydroxypyrimidines and hydroxyquinolines. Other examples will be recognized by those skilled in the art. All such tautomers and mixtures thereof are included in this invention.
The subject invention also includes isotopically-labeled compounds, which are identical to those recited in Formula I, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F and 36Cl, respectively. Compounds of Formula I of the present invention, prodrugs thereof, and pharmaceutically acceptable salts of said compounds and said prodrugs, and stereoisomers and diastereomeric mixtures of said compounds, prodrugs and salts, which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labeled compounds of the present invention, for example those into which radioactive isotopes such as 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds of Formula I of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the Schemes and /or in the Examples and Preparations below, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
The compounds of Formula I of this invention have asymmetric carbon atoms and therefore are enantiomers or diastereomers. Diasteromeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods known per se, for example, by chromatography and/or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diasteromeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. Enantiomers and diastereomers of this invention can also be prepared by utilizing suitable enantiomerically enriched starting materials, or by asymmetric or diastereoselective reactions to introduce asymmetric carbon atoms with the correct stereochemistry. All such isomers, including diastereomers, enantiomers and mixtures thereof are considered as part of this invention. Some of the compounds of this invention are acidic and they form a salt with a pharmaceutically acceptable cation. All such salts are within the scope of this invention and they can be prepared by conventional methods. For example, they can be prepared simply by contacting the acidic and basic entities, usually in a stoichiometric ratio, in either an aqueous, non-aqueous or partially aqueous medium, as appropriate. The salts are recovered either by filtration, by precipitation with a non-solvent followed by filtration, by evaporation of the solvent, or, in the case of aqueous solutions, by lyophilization, as appropriate.
The methods of this invention result in bone formation resulting in decreased fracture rates. This invention makes a significant contribution to the art by providing methods that increase bone formation resulting in prevention, retardation, and/or regression of osteoporosis and related bone disorders.
Other features and advantages will be apparent from the description and claims which describe the invention.
In general, the compounds of Formula I of this invention (hereinafter collectively referred to as xe2x80x9cthe compounds of this inventionxe2x80x9d) are made by processes which include processes analogous to those known in the chemical arts. These processes include methods which may require protection of remote functionality (e.g., primary amine, secondary amine, secondary alcohol, primary alcohol, carboxyl in Formula I precursors). The need for such protection will vary depending upon the nature of the remote functionality and the conditions of the preparation methods. The need for such protection is readily determined by one skilled in the art. The use of such protection/deprotection methods is also within the skill in the art. The term xe2x80x9cprotecting group,xe2x80x9d where used herein, refers to a radical which may be attached to a functional group on a substrate which is easily attached and easily removed without affecting other functional groups of the substrate and which prevents the protected functional group from being removed, altered or otherwise destroyed. For a general description of protecting groups and their use, see Greene, T. W.; Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd ed.; John Wiley and Sons Inc.: New York, 1991. The starting materials and reagents for the above described compounds are also readily available or can be easily synthesized by those skilled in the art using conventional methods of organic synthesis in light of this disclosure.
In general, compounds of Formula I are prepared by protection of the hydroxyl group of either racemic or (R)-hydroxymethyl-2-pyrrolidinone, followed by alkylation of the amide nitrogen with an alkyl halide which contains a suitably protected acid precursor or isostere (Scheme A). The term xe2x80x9cisostere,xe2x80x9d where used herein, refers to a functional group which, when used in place of another functional group, approximates the reactivity of the functional group which it replaces. In some cases, the alkyl halide must be further elaborated to install the suitably protected acid precursor or isostere (Scheme B1). The hydroxyl protecting group is removed, the alcohol oxidized to the aldehyde which is then reacted with the anion of a suitable keto-phosphonate (Scheme C). The resulting enone of formula 8 of Scheme E is then subjected to reduction of both the double bond and ketone to give the desired saturated alcohols of formula 9 of Scheme E. If desired, a diastereoselective reduction of the enone can be effected to give, for example, predominantly the 15-(R) isomer or the 15-(S) isomer. The carboxylic ester or precursor to an acid isostere (e.g., nitrile) is then converted into the appropriate acidic group (carboxylic acid, tetrazole, etc).
A preferred method for converting a nitrile into the desired tetrazole is treatment of the nitrile with dibutyltin oxide and trimethylsilylazide, in refluxing toluene (S. J. Wittenberger and B. G. Donner, J. Org. Chem. 1993, 58, 4139-4141, 1993). For a review of alternative preparations of tetrazoles see R. N. Butler, Tetrazoles, in Comprehensive Heterocyclic Chemistry; Potts, K. T. Ed.; Pergamon Press: Oxford, 1984, Vol. 5, pp 791-838. 
More specifically, compounds of Formula I are prepared by the following procedures. In the first general sequence, which begins with Scheme A, the hydroxyl group of 5-(R)-hydroxymethyl-2-pyrrolidinone (Aldrich Chemical, or prepared as described by Bruckner et al., Acta. Chim. Hung. Tomus, 21, 106 (1959)) is suitably protected (where PG is a suitable protecting group) by reaction of a compound of formula 1 in a reaction inert solvent. As used herein, the expressions xe2x80x9creaction inert solventxe2x80x9d and xe2x80x9cinert solventxe2x80x9d refer to a solvent or mixture of solvents which does not interact with starting materials, reagents, intermediates or products in a manner which adversely affects the yield of the desired product. In some cases herein, a list of preferred reaction inert solvents is described. However, any solvent which meets the above definition of reaction inert solvent for a particular reaction may be used in that reaction. All reactions are carried out in a reaction inert solvent unless specifically stated otherwise. Any standard alcohol protecting group may be utilized, including tetrahydropyranyl, trimethylsilyl, tert-butyl-dimethylsilyl, or benzyl. A preferred protecting group is tert-butyl-dimethylsilyl (TBS), which can be installed by standard methods as described in Greene, T. W.; Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd ed.; John Wiley and Sons Inc.: New York, 1991. It is preferred to treat 5-(R)-hydroxymethyl-2-pyrrolidinone in methylene chloride at 0xc2x0 C. with 0.1 eq of 4-dimethylaminopyridine, 1.1 eq. of tert-butyl-dimethylsilylchloride, and 2 eq. of imidazole (see, e.g., Tetrahedron Asymmetry, 7, 2113, (1996)). The amide nitrogen is alkylated with one of a variety of alkylating agents (halxe2x80x94CH2CH2xe2x80x94Xxe2x80x94Zxe2x80x94QP, where hal is a leaving group such as bromide or iodide, X and Z are as described in the Summary, and QP is a nitrile, carboxylic acid ester or other precursor to a carboxylic acid or acid isostere) to introduce the desired side chain. The amide nitrogen is first deprotonated with a suitable base. Preferred bases include sodium hexamethyldisilazide (also referred to herein as NaHMDS or NaN(SiMe3)2) or sodium hydride in a reaction inert solvent such as N,N-dimethylformamide (DMF), tetrahydrofuran (THF), 1,2-dimethoxyethane or 1,4-dioxane. A preferred solvent is DMF. The appropriate temperature range for anion formation is between xe2x88x9278xc2x0 C. and the temperature at which the solvent refluxes. A preferred temperature for this reaction is about 0xc2x0 C. After formation of the anion, the alkylating agent (halxe2x80x94CH2CH2xe2x80x94Xxe2x80x94Zxe2x80x94QP) is added and the solution is stirred at an appropriate temperature. The appropriate temperature range for alkylation is between xe2x88x9220xc2x0 C. and the temperature at which the solvent refluxes. The preferred temperature range for this reaction is between 0xc2x0 C. and 100xc2x0 C. Typical alkylating agents are primary, secondary, benzylic, propargyllic halides and primary, secondary, benzylic or propargyllic sulfonates. Preferred alkylating agents are alkyl bromides or alkyl iodides.
Many of the useful alkylating agents of the formula halxe2x80x94CH2CH2xe2x80x94Xxe2x80x94Zxe2x80x94QP are commercially available. For example, ethyl-7-bromoheptanoate and 7-bromoheptanonitrile may be obtained from Aldrich Chemical, P.O. Box 355, Milwaukee, Wis. 53201, USA. Numerous methods known to those skilled in the art exist for the synthesis of those and other desired alkylating agents used in the above Scheme (see, e.g., xe2x80x9cThe Chemistry of the Carbon-Halogen Bond,xe2x80x9d Ed. S. Patai, J. Wiley, New York, 1973 and/or xe2x80x9cThe Chemistry of Halides, Pseudo-Halides, and Azides,xe2x80x9d Eds. S. Patai and Z. Rappaport, J. Wiley, New York, 1983).
Alkyl halides are also prepared by halogenation of an alcohol or an alcohol derivative. Alkyl chlorides are typically prepared from the alcohols with reagents such as hydrogen chloride, thionyl chloride, phosphorous pentachloride, phosphorous oxychloride or triphenylphosphine/carbon tetrachloride in a reaction inert solvent. For the preparation of alkyl bromides the alcohol is commonly treated with reagents such as hydrogen bromide, phosphorous tribromide, triphenylphosphine/bromine or carbonyldiimidazole/allyl bromide in a reaction inert solvent. To prepare alkyl iodides, the alcohol is typically reacted with reagents such as triphenylphosphine/iodine/imidazole or hydrogen iodide in a reaction inert solvent. Alkyl chlorides are converted to the more reactive alkyl bromides or alkyl iodides by treatment with an inorganic salt such as sodium bromide, lithium bromide, sodium iodide or potassium iodide in a reaction inert solvent such as acetone or methyl ethyl ketone. Alkyl sulfonates are also used as electrophiles or are converted to alkyl halides. Sulfonates are prepared from the alcohol using a mild base such as triethylamine or pyridine and a sulfonyl chloride in a reaction inert solvent such a methylene chloride or diethyl ether. Conversion to the halide is accomplished by treatment of the alkyl sulfonate with an inorganic halide (sodium iodide, sodium bromide, potassium iodide, potassium bromide, lithium chloride, lithium bromide, etc) or a tetrabutylammonium halide in a reaction inert solvent.
Alkyl halides of the formula halxe2x80x94CH2CH2xe2x80x94Xxe2x80x94Zxe2x80x94QP where X is CH2 and Z is phenyl, thienyl or thiazolyl are also prepared as shown in Scheme B1. For example, propargyl alcohol is treated with a compound of formula 14 of Scheme B1 containing the suitably protected acid isostere (halxe2x80x94Zxe2x80x94QP), where the xe2x80x9chalxe2x80x94Zxe2x80x9d group is an aryl bromide, iodide or triflate, in the presence of copper (I) iodide; a palladium catalyst such as palladium chloride, bis(triphenylphosphine)palladium dichloride or tetrakis(triphenylphosphine) palladium(0); and an amine such as triethylamine, diisopropylamine or butylamine in a reaction inert solvent, preferably an aprotic solvent such as acetonitrile, at a temperature of about 0xc2x0 C. to about 100xc2x0 C. For additional references, see Tetrahedron, 40, 1433 (1984) and Org. Lett. 2, 12, 1729 (2000). The resulting alkynes are then converted to the corresponding alkanes via hydrogenation in the presence of a palladium or platinum catalyst in a reaction inert solvent such as methanol, ethanol and/or ethyl acetate at a temperature of about 0xc2x0 C. to about 50xc2x0 C. The alcohol portion of the molecule is replaced with a suitable leaving group such as bromide or iodide. For the preparation of alkyl bromides, the alcohol is commonly treated with reagents such as hydrogen bromide, phosphorous tribromide, triphenylphosphine/bromine or carbonyldiimidazole/allyl bromide. The use of carbonyldiimidazole/allyl bromide is preferred. To prepare alkyl iodides, the alcohol is typically reacted with a reagent such as triphenylphosphine/iodine/imidazole or hydrogen iodide in a reaction inert solvent. Alkyl chlorides are converted to the more reactive alkyl bromides or alkyl iodides by treatment with an inorganic salt such as sodium bromide, lithium bromide, sodium iodide or potassium iodide in a reaction inert solvent such as acetone or methyl ethyl ketone. Alkyl sulfonates can be used as electrophiles or are converted to alkyl halides. Alkyl sulfonates are prepared from the corresponding alcohol using a mild base such as triethylamine or pyridine and a sulfonyl chloride in a reaction inert solvent such as methylene chloride or diethyl ether. Conversion to the halide is accomplished by treating the alkyl sulfonate with an inorganic halide such as, for example, sodium iodide, sodium bromide, potassium iodide, potassium bromide, lithium chloride or lithium bromide in a reaction inert solvent. Conversion to the halide may also be accomplished by treating the alkyl sulfonate with an organic ammonium halide such as tetrabutylammonium halide in a reaction inert solvent. Alkyl chlorides are typically prepared from the alcohols with reagents such as hydrogen chloride, thionyl chloride, phosphorous pentachloride, phosphorous oxychloride, or triphenylphosphine/carbon tetrachloride. 
In some cases, as shown in Scheme B2, it is preferred to first alkylate with propargyl bromide or iodide, and then further elaborate to introduce the suitably protected acid precursor or isostere. For example, where the alkylating agent is propargyl bromide or iodide, compounds of Formula 3 of Scheme B2 are treated with compounds of Formula 14 of Scheme B2 containing the suitably protected acid precursor or isostere (halxe2x80x94Zxe2x80x94QP), where the xe2x80x9chalxe2x80x94Zxe2x80x9d group is an aryl bromide, iodide or triflate, in the presence of copper (I) iodide; a palladium catalyst such as palladium chloride, bis(triphenylphosphine)palladium dichloride or tetrakis(triphenylphosphine) palladium(0); and an amine such as triethylamine, diisopropylamine or butylamine in a reaction inert solvent, preferably an aprotic solvent such as acetonitrile, at a temperature of about 0xc2x0 C. to about 100xc2x0 C. For additional references see Tetrahedron, 40, 1433 (1984) and Org. Lett. 2,12, 1729 (2000). The resulting alkynes are then converted to the corresponding alkanes via hydrogenation in the presence of a palladium or platinum catalyst in a reaction inert solvent such as methanol, ethanol and/or ethyl acetate at a temperature of about 0xc2x0 C. to about 50xc2x0 C. 
Halo-arylesters and halo-arylnitriles of Formula 14 of Scheme B2 are prepared by methods known to those skilled in the art. For example, 2-bromo-4-(ethoxycarbonyl)thiazole is prepared according to the procedure described in J. Org. Chem. 61, 14, 4623, (1996); and 2-bromo-5-(ethoxycarbonyl)thiazole is prepared according to the procedure described in Helv. Chim. Acta, 25, 1073, (1942). Other halo-arylesters and halo-arylnitriles of Formula 14 of Scheme B2 which are useful in the procedures of this invention, such as, inter alia, ethyl-4-bromobenzoate and 4-bromobenzonitrile are commercially available. Ethyl-2-bromo-thiophene-5-carboxylate is prepared by esterification of commercially available 2-bromo-thiophene-5-carboxylic acid.
The alcohol protecting groups of compounds of Formula 2 of Scheme A or Formula 4 of Scheme B2 are then removed. For a general description of methods for deprotection of protected alcohols, see Greene, T. W.; Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd ed.; John Wiley and Sons Inc.: New York, 1991. Removal of the tert-butyl-dimethylsilyl group in compounds of Formula 2 and Formula 4 of Scheme B2 is preferably accomplished by treating the compound with tetrabutylammonium fluoride or trifluoroacetic acid in a reaction inert solvent, preferably in a suitable aprotic solvent at a temperature of about of xe2x88x9230xc2x0 C. to about ambient temperature. Where used herein, the term xe2x80x9cambient temperaturexe2x80x9d refers to the temperature of the immediate, unaltered surroundings of the reaction mixture. Ambient temperature is generally between 20xc2x0 C. and 25xc2x0 C. An especially preferred solvent is methylene chloride. A preferred temperature range is between 0xc2x0 C. to ambient temperature. Another preferred method to remove the TBS group is by treatment of the silyl ether with an aqueous solution of a mineral acid in a protic solvent. In this case, it is preferred that the silyl ether is treated with a 1N aqueous solution of hydrochloric acid in methanol at ambient temperature. Subsequent to deprotection, the alcohols are oxidized to the aldehyde by use of a modification of the Pfitzner Moffatt oxidation [K. E. Pfitzner and M. E. Moffatt, J. Am. Chem. Soc., 87, 5661 (1965)] which minimizes racemization by avoiding contact with water. For example, oxidation of the alcohol to the aldehyde is achieved by stirring the alcohol in a reaction inert solvent, preferably a hydrocarbon solvent such as toluene, xylene or, preferably, benzene, with dimethyl sulfoxide, a weak acid such as acetic acid or, preferably, pyridinium trifluoroacetate, and a diimide such as diethyl carbodiimide or, preferably, dimethylaminopropylethylcarbodiimide or, if desired, dimethylaminopropylethylcarbodiimide hydrochloride, at temperatures of about 0xc2x0 C. to about ambient temperature for about one to about four hours. Alternate methods to achieve oxidation while minimizing racemization of the asymmetric center adjacent to the resulting aldehyde are discussed in detail in Tetrahedron Letters, 41, 1359, (2000) and include the usual Pfitzner-Moffatt reaction, oxidation with chromium trioxide-pyridine complex [J. Org. Chem., 35, 4000 (1970)], oxidation with Dess-Martin reagent [J. Org. Chem. 48, 4155, (1983)] or oxidation with TEMPO-bleach [Tetrahedron Letters 33, 5029, (1992)].
The resulting aldehyde is preferably subjected without purification to a Horner-Wittig reaction with the sodium or lithium salt of a phosphonate of Formula 7 of Scheme C (R is lower alkyl, haloalkyl or aryl). The sodium or lithium salts are pre-formed by prior treatment of the phosphonates with a suitable base such as sodium hydride or NaN(SiMe3)2 in a suitable reaction inert solvent, preferably an aprotic ethereal solvent at a temperature of about 0xc2x0 C. to about 50xc2x0 C. A preferred solvent is THF and a preferred temperature is ambient temperature. A solution of the aldehyde is then added to the salt of the phosphonate in a reaction inert solvent, preferably an aprotic solvent at a temperature of about 0xc2x0 C. to about 50xc2x0 C. to give enones of Formula 8 of Scheme C. A preferred solvent is THF. A preferred temperature is ambient temperature. 
Methods for the preparation of phosphonates of Formula 7 of Scheme C1 can be found in U.S. Pat. Nos. 3,932,389; 4,177,346; Tetrahedron Lett., 30, 36, 4787-4790, (1989); and Angew.Chem., 108, 3, 366-369, (1996). In general, as shown in Scheme C1, the phosphonates of Formula 7 are prepared from reaction of the appropriately substituted arylacetic acid esters or the methoxymethyl amide of the arylacetic acid with th elithium reagent derived from a dialkyl methylphosphonate. These methods are also applicable to cycloalkylacetic esters and methoxymethylamides such as ethyl-cyclohexylacetate and ethyl-cyclopentylacetate. The aryl- and cycloalkyl-acetic acid esters are prepared by esterification of the corresponding acetic acid by methods known to those skilled in the art. The methoxymethylamides are prepared by a standard amide bond forming reaction between the corresponding acetic acid and methoxymethyl amine. Preferably the coupling of the amine with the carboxylic acid is carried out in a reaction inert solvent such as dichloromethane or DMF by a coupling reagent such as 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (EDC) or 1,3-dicyclohexylcarbodiimide (DCC) in the presence of an acid activating agent such as 1-hydroxybenzotriazole hydrate (HOBT) to generate the methoxymethyl amide. In the case where the amine is present as the hydrochloride salt, it is preferable to add one equivalent of a suitable base such as triethylamine to the reaction mixture. Alternatively, coupling of the amine with the carboxylic acid is effected with a coupling reagent such as benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP) in a reaction inert solvent such as methanol. Such coupling reactions are generally conducted at temperatures of about xe2x88x9230xc2x0 C. to about 80xc2x0 C., preferably about 0xc2x0 C. to about 25xc2x0 C. For a discussion of other conditions used for amide couplings, see HeubenWeyl, Vol. XV, part 11, E. Wunsch, Ed., George Theime Verlag, 1974, Stuttgart. 
The requisite arylacetic acids and esters of Formula 6 of Scheme C1 are commercially available or are prepared by methods well known to those skilled in the art. As shown in Scheme C2, many aryl and heteroaryl substituted aryl acetic acids are prepared by Suzuki couplings of the appropriate arylboronic acids or arylboronate esters with the desired aryl halides (for a review of the Suzuki coupling reaction see A. R. Martin and Y. Yang in Acta Chem. Scand. 1993, 47, 221 or J. Am. Chem. Soc., 2000, 122, 17, 4020). For example, the 3-pinacolboronate ester of ethyl-3-bromophenylacetate is prepared using the method described by Masuda et al. in J. Org. Chem., 65, 164 (2000). Said 3-pinacolboronate ester of ethyl-3-bromophenylacetate is then coupled with the desired aryl halide to give the desired 3-aryl-phenylacetic acid (see Synlett., 6, 829 (2000)). Hydroxy substituted aryl acetic esters are alkylated with alkyl halides and benzylic halides by methods well known to those skilled in the art. 
For a review of the preparation of diaryl ethers, see Angew. Chem., Int. Ed., 38, 16, 2345, (1999). Aryl acetic acids substituted with an alkylether linkage are prepared using Mitsunobu conditions (for a review see Synthesis, 1, (1981)). Typically, the coupling between a phenolic component and a benzylic alcohol is achieved by addition of triphenylphosphine and diethyl azodicarboxylate or diisopropyl azodicarboxylate in a reaction inert solvent such as methylene chloride or THF.
Alternatively, phosphonates of Formula 7 of Scheme D are prepared as shown in Scheme D. In general, triethylphosphite is added slowly to epibromo- or epichloro-hydrin (10) at a temperature of about 135xc2x0 C. As the triethylphosphite is added, the temperature drops to about 105xc2x0 C. The reaction mixture is refluxed overnight and the product, a compound of formula 11, is isolated by vacuum distillation (see Phosphorus, Sulfur Silicon Relat. Elem., 165, 71 (1992) or U.S. Pat. No. 2,627,521). The required Grignard solutions are prepared from the appropriate aryl halides according to procedures well known to those skilled in the art in a reaction inert solvent, preferably an ethereal solvent such as THF and cooled to approximately xe2x88x9230xc2x0 C. Catalytic copper (I) iodide is added followed by addition of the epoxide of Formula 11 [Phosphorus, Sulfur Silicon Relat. Elem., 105, 45 (1995)]. The requisite aryl halides (e.g., 3-bromo-biphenyl) are commercially available or are prepared by methods well known to those skilled in the art.
The resulting alcohols are then oxidized, preferably using a Swern oxidation [Synthesis, pp.165-185, (1981)] or Dess-Martin reagent [J. Org. Chem. 48, 4155, (1983)]. Alternative oxidation procedures such as Pfitzner-Moffatt reaction, chromium trioxide-pyridine complex [R. Ratcliffe, et al., J. Org. Chem., 35, 4000 (1970)], TEMPO-bleach [Tet. Lett. 33, 5029, (1992)], Jones oxidation, Manganese dioxide, pyridiniumchlorochromate or pyridinium dichromate may also be utilized to prepare keto-phosphonates of Formula 7 of Scheme D. 
An enone of Formula 8 of Scheme E (which may also be prepared as shown in Scheme C) is reduced to a mixture of alcohol diastereomers of Formula 9 of Scheme E by methods well known to those skilled in the art. In general, the double bond of the enone is first reduced by catalytic hydrogenation. It is preferred that the double bond is reduced by hydrogenation over a noble metal catalyst such as palladium on carbon or platinum oxide in a reaction inert solvent such as ethyl acetate, methanol or ethanol at ambient temperature to about the reflux temperature of the solvent being used under 1-4 atmospheres of hydrogen. The resulting ketone is then treated with a reducing agent, preferably sodium borohydride, in a protic solvent, preferably ethanol or methanol, to give alcohols of Formula 9 of Scheme E. Other selective reduction reagents well known to those skilled in the art which will reduce the ketone but no other groups, e.g. zinc borohydride or lithium triethylborohydride may be employed with equal facility. The temperature selection will be based upon the activity of the reducing agent and will preferably be between about 0xc2x0 C. to ambient temperature. If desired, the mixture of alcohols of Formula 9 may be separated by preparative chromatography or HPLC to give the desired 15-(R) diastereomer.
In an alternative sequence shown in Scheme E, an enone of Formula 8 of Scheme E is first treated with a hydride reducing agent in the presence of a chiral catalyst. Where used herein, the term xe2x80x9chydride reducing agentxe2x80x9d refers to compounds which are able to reduce a compound having a higher oxidation state by transferring hydrogen to the compound. A preferred hydride reducing agent is catecholborane. A preferred chiral catalyst for performing such reactions enantioselectively is (R)-2-methyl-CBS-oxazaborolidine reagent (Aldrich Chemical Co.) (see the method described in Eur. J. Org. Chem., 2655 (1999)). The reduction is carried out in a reaction inert solvent, preferably an aprotic solvent such as methylene chloride, at a temperature of about xe2x88x92100xc2x0 C. to ambient temperature. A preferred temperature for this reaction is about xe2x88x9240xc2x0 C. Alternative methods and catalysts which are utilized to effect stereoselective reduction of the enone carbonyl are described in J. Am. Chem. Soc., 117, 2675, (1995); J. Am. Chem. Soc., 101, 5843, (1979); Tett. Lett., 31, 611, (1990); U.S. Pat. No. 6,037,505; and Angew. Chem. Int. Ed., 37, 1986, (1998). The double bond of the allylic alcohol is then reduced. It is preferred that the double bond is reduced by hydrogenation over a noble metal catalyst such as palladium on carbon or platinum oxide in a reaction inert solvent such as ethyl acetate, methanol or ethanol at ambient temperature to the reflux temperature of the solvent being used under 1-4 atmospheres of hydrogen. 
An alternative procedure for the preparation of compounds of formula 9 of Scheme F is shown in Scheme F. In general, tetrahydro-pyrrolizine-3,5-dione (the compound of formula 12 of Scheme F) is prepared as described in U.S. Pat. No. 4,663,464 or J.Med.Chem. 30; 3; 498-503; (1987). The compound of Formula 12 of Scheme F is then dissolved in a reaction inert solvent, preferably an aprotic solvent at a suitable temperature. It is preferred that said compound is dissolved in methylene chloride at about 0xc2x0 C. The reaction mixture is then treated with the appropriate Grignard reagent (for additional references on addition of Grignard reagents to Formula 12 of Scheme F, see Synth.Commun., 18, 1, 37-44, (1988);
Helv.Chim.Acta, 70, 2003-2010, (1987)). The reaction may be warmed to ambient temperature to effect complete reaction. The resulting ketone is then treated with a reducing agent, preferably sodium borohydride in a protic solvent, preferable ethanol or methanol. Other selective reducing reagents which will reduce the ketone but no other groups, e.g., zinc borohydride or lithium triethylborohydride, can be employed with equal facility. The temperature selection will be based upon the activity of the reducing agent, preferably from about 0xc2x0 C. to ambient temperature. The resulting hydroxyl group is then suitably protected. Standard alcohol protecting groups such as tetrahydropyranyl, trimethylsilyl, tert-butyl-dimethylsilyl or benzyl may be utilized. A preferred protecting group is tert-butyl-dimethylsilyl which is installed by standard methods as described in Greene, T. W.; Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd ed.; John Wiley and Sons Inc.: New York, 1991. Preferred conditions for this reaction include treating the alcohol in DMF at ambient temperature with 0.1 eq of 4-dimethylaminopyridine, 1.1 eq. of tert-butyl-dimethylsilylchloride and 2 eq. of imidazole.
The resulting compound of Formula 13 of Scheme F is then alkylated on nitrogen with one of a variety of alkylating agents of the formula halxe2x80x94CH2CH2xe2x80x94Xxe2x80x94QP to introduce the desired side chain. The amide nitrogen is first deprotonated with a suitable base in a reaction inert solvent. Preferred bases for this reaction include NaN(SiMe3)2 or sodium hydride in a solvent such as DMF, tetrahydrofuran, dimethoxyethane or dioxane. An especially preferred solvent is DMF. The appropriate temperature range for anion formation is between xe2x88x9278xc2x0 C. and about the temperature at which the solvent refluxes. It is preferred that the reaction is conducted at ambient temperature. After formation of the anion, the alkylating agent of the formula halxe2x80x94CH2CH2xe2x80x94Xxe2x80x94QP is added, and the solution is stirred at a temperature between xe2x88x9220xc2x0 C. to about the temperature at which the solvent refluxes. A preferred temperature is between ambient temperature and 100xc2x0 C. Typical alkylating agents include primary halides and primary sulfonates. Preferably, an alkyl bromide or alkyl iodide is used. The alcohol protecting group is then removed by methods well known to those skilled in the art (see Greene, T. W.; Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd ed.; John Wiley and Sons Inc.: New York, 1991) to produce compounds of Formula 9. 
Compounds of formula 9 of Scheme F are converted to compounds of Formula I by methods well known to those skilled in the art. In cases where the QP group is a carboxylic ester, either acidic or basic aqueous hydrolysis conditions may be utilized. Typically, lower alkyl esters are hydrolyzed by base catalyzed hydrolysis in a reaction inert solvent at ambient temperature to about the reflux temperature of the solvent being used. Preferably the lower alkyl ester is hydrolyzed with aqueous 1 N sodium hydroxide in methanol at a suitable temperature, preferably at ambient temperature. When QP is a benzyl ester or a t-butyl ester, standard deprotection methods are utilized as described in Greene, T. W.; Wuts, P. G. M., Protective Groups in Organic-Synthesis, 2nd ed.; John Wiley and Sons Inc.: New York, 1991. When QP is a nitrile and not a protected carboxylic acid, a preferred method for preparation of the tetrazole is treatment of the nitrile with dibutyltin oxide and trimethylsilylazide in refluxing toluene (S. J. Wittenberger and B. G. Donner, J. Org. Chem. 1993, 58, 4139-4141, 1993). For a review of alternative preparations of tetrazoles see R. N. Butler, Tetrazoles, In comprehensive Heterocyclic Chemistry; Potts, K. T. Ed.; Pergamon Press: Oxford, 1984, Vol. 5, p 791-838.
The EP4 receptor selective agonists of Formula I of this invention are all adapted to therapeutic use as agents that stimulate bone formation and increase bone mass in vertebrates, e.g., mammals, and particularly humans. Since bone formation is closely related to the development of osteoporosis and bone related disorders, the agonists used in the methods of this invention, by virtue of their action on bone, prevent, arrest and/or regress osteoporosis.
The utility of the EP4.selective agonists of Formula I of the present invention as medical agents in the treatment of conditions which present with low bone mass (e.g., osteoporosis) in vertebrates, e.g., mammals (especially humans and particularly female humans) is demonstrated by the activity of those agonists in conventional assays, including a cyclic AMP assay, an in vivo assay and a fracture healing assay, all of which are described below. Such assays also provide a means whereby the activities of the EP4 selective agonists of Formula I of this invention can be compared to each other and with the activities of other known compounds and compositions. The results of these comparisons are useful for determining dosage levels in a vertebrates, e.g., mammals, including humans, for the treatment of such diseases.
The activity of anabolic bone agents in stimulating bone formation and increasing bone mass can be tested in intact male or female rats, sex hormone deficient male (orchidectomy) or female (ovariectomy) rats.
Male or female rats at different ages (such as 3 months of age) can be used in the study. The rats are either intact or castrated (ovariectomized or orchidectomized), and subcutaneously injected or gavaged with a compound of Formula I of this invention at different doses (such as 1, 3, or 10 mg/kg/day) for 30 days. In the castrated rats, treatment is started on the next day after surgery (for the purpose of preventing bone loss) or at the time bone loss has already occured (for the purpose of restoring bone mass). During the study, all rats are allowed free access to water and a pelleted commercial diet (Teklad Rodent Diet #8064, Harlan Teklad, Madison, Wis.) containing 1.46% calcium, 0.99% phosphorus and 4.96 IU/g of Vitamin D3. All rats are given subcutaneous injections of 10 mg/kg calcein on days 12 and 2 before sacrifice. The rats are sacrificed. The following endpoints are determined:
Femoral Bone Mineral Measurements
The right femur from each rat is removed at autopsy and scanned using dual energy X-ray absorptiometry (DXA, QDR 1000/W, Hologic Inc., Waltham, Mass.) equipped with xe2x80x9cRegional High Resolution Scanxe2x80x9d software (Hologic Inc., Waltham, Mass.). The scan field size is 5.08xc3x971.902 cm, resolution is 0.0254xc3x970.0127 cm and scan speed is 7.25 mm/second. The femoral scan images are analyzed and bone area, bone mineral content (BMC), and bone mineral density (BMD) of whole femora (WF), distal femoral metaphyses (DFM), femoral shaft (FS), and proximal femora (PF) are determined.
Tibial Bone Histomorphometric Analyses
The right tibia is removed at autopsy, dissected free of muscle, and cut into three parts. The proximal tibia and the tibial shaft are fixed in 70% ethanol, dehydrated in graded concentrations of ethanol, defatted in acetone, then embedded in methyl methacrylate (Eastman Organic Chemicals, Rochester, N.Y.).
Frontal sections of proximal tibial metaphyses at 4 and 10 xcexcm thickness are cut using a Reichert-Jung Polycut S microtome. The 4 xcexcm sections are stained with modified Masson""s Trichrome stain while the 10 xcexcm sections remain unstained. One 4 xcexcm and one 10 xcexcm section from each rat are used for cancellous bone histomorphometry.
Cross sections of tibial shaft at 10 xcexcm thickness are cut using a Reichert-Jung Polycut S microtome. These sections are used for cortical bone histomorphometric analysis.
Cancellous Bone Histomorphometry
A Bioquant OS/2 histomorphometry system (RandM Biometrics, Inc., Nashville, Tenn.) is used for the static and dynamic histomorphometric measurements of the secondary spongiosa of the proximal tibial metaphyses between 1.2 and 3.6 mm distal to the growth plate-epiphyseal junction. The first 1.2 mm of the tibial metaphyseal region needs to be omitted in order to restrict measurements to the secondary spongiosa. The 4 xcexcm sections are used to determine indices related to bone volume, bone structure, and bone resorption, while the 10 xcexcm sections are used to determine indices related to bone formation and bone turnover.
I) Measurements and Calculations Related to Trabecular Bone Volume and Structure:
(1) Total metaphyseal area (TV, mm2): metaphyseal area between 1.2 and 3.6 mm distal to the growth plate-epiphyseal junction. (2) Trabecular bone area (BV, mm2): total area of trabeculae within TV. (3) Trabecular bone perimeter (BS, mm): the length of total perimeter of trabeculae. (4) Trabecular bone volume (BV/TV, %): BV/TVxc3x97100. (5) Trabecular bone number (TBN, #/mm): 1.199/2xc3x97BS/TV. (6) Trabecular bone thickness (TBT, xcexcm): (2000/1.199)xc3x97(BV/BS). (7) Trabecular bone separation (TBS, xcexcm): (2000xc3x971.199)xc3x97(TVxe2x88x92BV).
II) Measurements and Calculations Related to Bone Resorption
(1) Osteoclast number (OCN, #): total number of osteoclast within total metaphyseal area. (2) Osteoclast perimeter (OCP, mm): length of trabecular perimeter covered by osteoclast. (3) Osteoclast number/mm (OCN/mm, #/mm): OCN/BS. (4) Percent osteoclast perimeter (% OCP, %): OCP/BSxc3x97100.
III) Measurements and Calculations Related to Bone Formation and Turnover
(1) Single-calcein labeled perimeter (SLS, mm): total length of trabecular perimeter labeled with one calcein label. (2) Double-calcein labeled perimeter (DLS, mm): total length of trabecular perimeter labeled with two calcein labels. (3) Inter-labeled width (ILW, xcexcm): average distance between two calcein labels. (4) Percent mineralizing perimeter (PMS, %): (SLS/2+DLS)/BSxc3x97100. (5) Mineral apposition rate (MAR, xcexcm/day): ILW/label interval. (6) Bone formation rate/surface ref. (BFR/BS, xcexcm2/d/xcexcm): (SLS/2+DLS)xc3x97MAR/BS. (7) Bone turnover rate (BTR, %/y): (SLS/2+DLS)xc3x97MAR/BVxc3x97100.
Cortical Bone Histomorphometry
A Bioquant OS/2 histomorphometry system (RandM Biometrics, Inc., Nashville, Tenn.) is used for the static and dynamic histomorphometric measurements of tibial shaft cortical bone. Total tissue area, marrow cavity area, periosteal perimeter, endocortical perimeter, single labeled perimeter, double labeled perimeter, and interlabeled width on both periosteal and endocortical surface are measured, and cortical bone area (total tissue areaxe2x80x94marrow cavity area), percent cortical bone area (cortical area/total tissue areaxc3x97100), percent marrow area (marrow cavity area/total tissue areaxc3x97100), periosteal and endocortical percent labeled perimeter [(single labeled perimeter/2+double labeled perimeter)/total perimeterxc3x97100], mineral apposition rate (interlabeled width/intervals), and bone formation rate [mineral apposition ratexc3x97[(single labeled perimeter/2+double labeled perimeter)/total perimeter] are calculated.
Statistics
Statistics can be calculated using StatView 4.0 packages (Abacus Concepts, Inc., Berkeley, Calif.). The analysis of variance (ANOVA) test followed by Fisher""s PLSD (Stat View, Abacus Concepts Inc., 1918 Bonita Ave, Berkeley, Calif. 94704-1014) are used to compare the differences between groups.
The full length coding sequence for the EP1 receptor is made as disclosed in Funk et al., Journal of Biological Chemistry, 1993, 268, 26767-26772. The full length coding sequence for the EP2 receptor is made as disclosed in Regan et al., Molecular Pharmacology, 1994, 46, 213-220. The full length coding sequence for the EP3 receptor is made as disclosed in Regan et al., British Journal of Pharmacology, 1994, 112, 377-385. The full length coding sequence for the EP4 receptor is made as disclosed in Bastien, Journal of Biological Chemistry, 1994, 269, 11873-11877. These full length receptors are used to prepare 293S cells expressing the EP1, EP2, EP3 or EP4 receptors.
293S cells expressing either the human EP1, EP2, EP3 or EP4 prostaglandin E2 receptors are generated according to methods known to those skilled in the art. Typically, PCR (polymerase chain reaction) primers corresponding to the 5xe2x80x2 and 3xe2x80x2 ends of the published full length receptor are made according to the well known methods disclosed above and are used in an RT-PCR reaction using the total RNA from human kidney (for EP1), human lung (for EP2), human lung (for EP3) or human lymphocytes (for EP4) as a source. PCR products are cloned by the TA overhang method into pCR2.1 (Invitrogen, Carlsbad, Calif.) and identity of the cloned receptor is confirmed by DNA sequencing.
293S cells (Mayo, Dept. of Biochemistry, Northwestern Univ.) are transfected with the cloned receptor in pcDNA3 by electroporation. Stable cell lines expressing the receptor are established following selection of transfected cells with G418.
Clonal cell lines expressing the maximal number of receptors are chosen following a whole cell 3H-PGE2 binding assay using unlabeled PGE2 as a competitor.
Fracture Technique
Sprage-Dawley rats at 3 months of age are anesthetized with Ketamine. A 1 cm incision is made on the anteromedial aspect of the proximal part of the right tibia or femur. The following describes the tibial surgical technique. The incision is carried through to the bone, and a 1 mm hole is drilled 4 mm proximal to the distal aspect of the tibial tuberosity 2 mm medial to the anterior ridge. Intramedullary nailing is performed with a 0.8 mm stainless steel tube (maximum load 36.3 N, maximum stiffness 61.8 N/mm, tested under the same conditions as the bones). No reaming of the medullary canal is performed. A standardized closed fracture is produced 2 mm above the tibiofibular junction by three-point bending using specially designed adjustable forceps with blunt jaws. To minimize soft tissue damage, care is taken not to displace the fracture. The skin is closed with monofilament nylon sutures. The operation is performed under sterile conditions. Radiographs of all fractures are taken immediately after nailing, and rats with fractures outside the specified diaphyseal area or with displaced nails are excluded. The remaining animals are divided randomly into the following groups with 10-12 animals per each subgroup per time point for testing the fracture healing. The first group receives daily gavage of vehicle (water: 100% ethanol=95:5) at 1 ml/rat, while the others receive daily gavage from 0.01 to 100 mg/kg/day of the compound to be tested (1 ml/rat) for 10, 20, 40 and 80 days.
At 10, 20, 40 and 80 days, 10-12 rats from each group are anesthetized with Ketamine and sacrificed by exsanguination. Both tibiofibular bones are removed by dissection and all soft tissue is stripped. Bones from 5-6 rats for each group are stored in 70% ethanol for histological analysis, and bones from another 5-6 rats for each group are stored in a buffered Ringer""s solution (+4xc2x0 C., pH 7.4) for radiographs and biomechanical testing which is performed.
Histological Analysis
The methods for histologic analysis of fractured bone have been previously published by Mosekilde and Bak (The Effects of Growth Hormone on Fracture Healing in Rats: A Histological Description. Bone, 14:19-27, 1993). Briefly, the fracture site is sawed 8 mm to each side of the fracture line, embedded undecalcified in methymethacrylate, and cut frontals sections on a Reichert-Jung Polycut microtome in 8 xcexcm thick. Masson-Trichrome stained mid-frontal sections (including both tibia and fibula) are used for visualization of the cellullar and tissue response to fracture healing with and without treatment. Sirius red stained sections are used to demonstrate the characterisitics of the callus structure and to differentiate between woven bone and lamellar bone at the fracture site. The following measurements are performed: (1) fracture gapxe2x80x94measured as the shortest distance between the cortical bone ends in the fracture, (2) callus length and callus diameter, (3) total bone volume area of callus, (4) bony tissue per tissue area inside the callus area, (5) fibrous tissue in the callus, and (6) cartilage area in the callus.
Biomechanical Analysis
The methods for biomechanical analysis have been previously published by Bak and Andreassen (The Effects of Aging on Fracture Healing in Rats. Calcif Tissue Int 45:292-297, 1989). Briefly, radiographs of all fractures are taken prior to the biomechanical test. The mechanical properties of the healing fractures are analyzed by a destructive three- or four-point bending procedure. Maximum load, stiffness, energy at maximum load, deflection at maximum load, and maximum stress are determined.
Fracture Technique
Female or male beagle dogs at approximately 2 years of age are used under anesthesia in the study. Transverse radial fractures are produced by slow continuous loading in three-point bending as described by Lenehan et al. (Lenehan, T. M.; Balligand, M.; Nunamaker, D. M.; Wood, F. E.: Effects of EHDP on Fracture Healing in Dogs. J Orthop Res 3:499-507; 1985). A wire is pulled through the fracture site to ensure complete anatomical disruption of the bone. Thereafter, local delivery of the compound to be tested to the fracture site is achieved by slow release of compound delivered by slow release pellets or by administration of the compound in a suitable formulation such as a paste gel solution or suspension for 10, 15, or 20 weeks.
Histological Analysis
The methods for histologic analysis of fractured bone have been previously published by Peter et al. (Peter, C. P.; Cook, W. O.; Nunamaker, D. M.; Provost, M. T.; Seedor, J. G.; Rodan, G. A. Effects of alendronate on fracture healing and bone remodeling in dogs. J. Orthop. Res. 14:74-70, 1996) and Mosekilde and Bak (The Effects of Growth Hormone on Fracture Healing in Rats: A Histological Description. Bone, 14:19-27, 1993). Briefly, after sacrifice, the fracture site is sawed 3 cm to each side of the fracture line, embedded undecalcified in methymethacrylate, and cut on a Reichert-Jung Polycut microtome in 8 xcexcm thick of frontal sections. Masson-Trichrome stained mid-frontal sections (including both tibia and fibula) are used for visualization of the cellullar and tissue response to fracture healing with and without treatment. Sirius red stained sections are used to demonstrate the characterisitics of the callus structure and to differentiate between woven bone and lamellar bone at the fracture site. The following measurements are performed: (1) fracture gapxe2x80x94measured as the shortest distance between the cortical bone ends in the fracture, (2) callus length and callus diameter, (3) total bone volume area of callus, (4) bony tissue per tissue area inside the callus area, (5) fibrous tissue in the callus and (6) cartilage area in the callus.
Biomechanical Analysis
The methods for biomechanical analysis have been previously published by Bak and Andreassen (The Effects of Aging on Fracture Healing in Rats. Calcif Tissue Int 45:292-297, 1989) and Peter et al. (Peter, C. P.; Cook, W. O.; Nunamaker, D. M.; Provost, M. T.; Seedor, J. G.; Rodan, G. A. Effects of Alendronate On Fracture Healing And Bone Remodeling In Dogs. J. Orthop. Res. 14:74-70, 1996). Briefly, radiographs of all fractures are taken prior to the biomechanical test. The mechanical properties of the healing fractures are analyzed by a destructive three- or four-point bending procedures. Maximum load, stiffness, energy at maximum load, deflection at maximum load, and maximum stress are determined.
The role of a prostaglandin agonist in kidney regeneration is investigated by the ability of Prostaglandin E2 (PGE2) or a prostaglandin agonist to induce the expression of Bone Morphogenetic Protein 7 (BMP-7) in wild type 293S cells and in 293S cells transfected with EP2.
Methods
293S and EP2 293S cells are grown in Dulbecco""s Modified Egale medium (DMEM, Gibco, BRL; Gaithersburg, Md.). One day prior to treatment with PGE2 or a prostaglandin agonist, cells are plated at a density of 1.5xc3x97106 cells /10 cm dish. Generally about 16 to 24 hours later the cell monolayer is washed once with OptiMEM (Gibco, BRL; Gaithersburg, Md.) followed by the addition of 10 ml OptiMEM/dish in the presence and absense of vehicle (DMSO), PGE2 (10xe2x88x926M) or a prostaglandin agonist (10xe2x88x926M). Cells are harvested and RNA is extracted at 8, 16 and 24 hours. Northern blot analysis of total RNA (20 mg/lane) is carried out by probing the blots with 32P-labeled BMP-7 probe. The blots are normalized for RNA loading by hybridization with 32P-labeled 18s ribosomal RNA probe. PGE2 and prostaglandin agonists induce the expression of BMP-7 in the EP2 293S cells in a time dependent manner. Such induction of expression is generally not observed in the parental cell line. Given the known role of BMP-7 in kidney regeneration and the ability of an prostaglandin agonist to induce BMP-7 expression in 293S kidney cells in a time and receptor specific manner indicates a role for prostaglandin agonist in kidney regeneration.
Those skilled in the art will recognize that anti-resorptive agents (for example progestins, polyphosphonates, bisphosphonate(s), estrogen agonists/antagonists, estrogen, estrogen/progestin combinations, Premarin(copyright), estrone, estriol or 17xcex1- or 17xcex2-ethynyl estradiol) may be used in conjunction with the compounds of this invention.
Exemplary progestins are available from commercial sources and include: algestone acetophenide, altrenogest, amadinone acetate, anagestone acetate, chlormadinone acetate, cingestol, clogestone acetate, clomegestone acetate, delmadinone acetate, desogestrel, dimethisterone, dydrogesterone, ethynerone, ethynodiol diacetate, etonogestrel, flurogestone acetate, gestaclone, gestodene, gestonorone caproate, gestrinone, haloprogesterone, hydroxyprogesterone caproate, levonorgestrel, lynestrenol, medrogestone, medroxyprogesterone acetate, melengestrol acetate, methynodiol diacetate, norethindrone, norethindrone acetate, norethynodrel, norgestimate, norgestomet, norgestrel, oxogestone phenpropionate, progesterone, quingestanol acetate, quingestrone, and tigestol.
Preferred progestins are medroxyprogestrone, norethindrone and norethynodrel.
Exemplary bone resorption inhibiting polyphosphonates include polyphosphonates of the type disclosed in U.S. Pat. No. 3,683,080, the disclosure of which is incorporated herein by reference. Preferred polyphosphonates are geminal diphosphonates (also referred to as bis-phosphonates). Tiludronate disodium is an especially preferred polyphosphonate. Ibandronic acid is an especially preferred polyphosphonate. Alendronate is an especially preferred polyphosphonate. Zoledronic acid is an especially preferred polyphosphonate. Other preferred polyphosphonates are 6-amino-1-hydroxy-hexylidene-bisphosphonic acid and 1-hydroxy-3(methylpentylamino)-propylidene-bisphosphonic acid. The polyphosphonates may be administered in the form of the acid, or of a soluble alkali metal salt or alkaline earth metal salt. Hydrolyzable esters of the polyphosphonates are likewise included. Specific examples include ethane-1-hydroxy 1,1-diphosphonic acid, methane diphosphonic acid, pentane-1-hydroxy-1,1-diphosphonic acid, methane dichloro diphosphonic acid, methane hydroxy diphosphonic acid, ethane-1-amino-1,1-diphosphonic acid, ethane-2-amino-1,1-diphosphonic acid, propane-3-amino-1-hydroxy-1,1-diphosphonic acid, propane-N,N-dimethyl-3-amino-1-hydroxy-1,1-diphosphonic acid, propane-3,3-dimethyl-3-amino-1-hydroxy-1,1-diphosphonic acid, phenyl amino methane diphosphonic acid,N,N-dimethylamino methane diphosphonic acid, N(2-hydroxyethyl) amino methane diphosphonic acid, butane-4-amino-1-hydroxy-1,1-diphosphonic acid, pentane-5-amino-1-hydroxy-1,1-diphosphonic acid, hexane-6-amino-1-hydroxy-1,1-diphosphonic acid and pharmaceutically acceptable esters and salts thereof.
In particular, the compounds of this invention may be combined with a mammalian estrogen agonist/antagonist. Any estrogen agonist/antagonist may be used as the second compound of this invention. The term estrogen agonist/antagonist refers to compounds which bind with the estrogen receptor, inhibit bone turnover and/or prevent bone loss. In particular, estrogen agonists are herein defined as chemical compounds capable of binding to the estrogen receptor sites in mammalian tissue, and mimicking the actions of estrogen in one or more tissue. Estrogen antagonists are herein defined as chemical compounds capable of binding to the estrogen receptor sites in mammalian tissue, and blocking the actions of estrogen in one or more tissues. Such activities are readily determined by those skilled in the art of standard assays including estrogen receptor binding assays, standard bone histomorphometric and densitometer methods, and Eriksen E. F. et al., Bone Histomorphometry, Raven Press, New York, 1994, pages 1-74; Grier S. J. et. al., The Use of Dual-Energy X-Ray Absorptiometry In Animals, Inv. Radiol., 1996, 31(1):50-62; Wahner H. W. and Fogelman I., The Evaluation of Osteoporosis: Dual Energy X-Ray Absorptiometry in Clinical Practice., Martin Dunitz Ltd., London 1994, pages 1-296). A variety of these compounds are described and referenced below.
A preferred estrogen agonist/antagonist is droloxifene: (phenol, 3-(1-(4-(2-(dimethylamino)ethoxy)phenyl)-2-phenyl-1-butenyl)-, (E)-) and related compounds which are disclosed in U.S. Pat. No. 5,047,431, the disclosure of which is incorporated herein by reference.
Another preferred estrogen agonist/antagonist is 3-(4-(1,2-diphenyl-but-1-enyl)-phenyl)-acrylic acid, which is disclosed in Willson et al., Endocrinology, 1997, 138, 3901-3911.
Another preferred estrogen agonist/antagonist is tamoxifen: (ethanamine,2-(-4-(1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethyl, (Z)-2-, 2-hydroxy-1,2,3-propanetricarboxylate(1:1)) and related compounds which are disclosed in U.S. Pat. No. 4,536,516, the disclosure of which is incorporated herein by reference.
Another related compound is 4-hydroxy tamoxifen which is disclosed in U.S. Pat. No. 4,623,660, the disclosure of which is incorporated herein by reference.
A preferred estrogen agonist/antagonist is raloxifene: (methanone, (6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl)(4-(2-(1-piperidinyl)ethoxy)phenyl)hydrochloride) which is disclosed in U.S. Pat. No. 4,418,068, the disclosure of which is incorporated herein by reference.
Another preferred estrogen agonist/antagonist is toremifene: (ethanamine, 2-(4-(4-chloro-1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethyl-, (Z)-, 2-hydroxy-1,2,3-propanetricarboxylate (1:1) which is disclosed in U.S. Pat. No. 4,996,225, the disclosure of which is incorporated herein by reference.
Another preferred estrogen agonist/antagonist is centchroman: 1-(2-((4-(-methoxy-2,2, dimethyl-3-phenyl-chroman-4-yl)-phenoxy)-ethyl)-pyrrolidine, which is disclosed in U.S. Pat. No. 3,822,287, the disclosure of which is incorporated herein by reference. Also preferred is levormeloxifene.
Another preferred estrogen agonist/antagonist is idoxifene: (E)-1-(2-(4-(1-(4-iodo-phenyl)-2-phenyl-but-1-enyl)-phenoxy)-ethyl)-pyrrolidinone, which is disclosed in U.S. Pat. No. 4,839,155, the disclosure of which is incorporated herein by reference.
Another preferred estrogen agonist/antagonist is 2-(4-methoxy-phenyl)-3-[4-(2-piperidin-1-yl-ethoxy)-phenoxy]-benzo[b]thiophen-6-ol which is disclosed in U.S. Pat. No. 5,488,058, the disclosure of which is incorporated herein by reference.
Another preferred estrogen agonist/antagonist is 6-(4-hydroxy-phenyl)-5-(4-(2-piperidin-1-yl-ethoxy)-benzyl)-naphthalen-2-ol which is disclosed in U.S. Pat. No. 5,484,795, the disclosure of which is incorporated herein by reference.
Another preferred estrogen agonist/antagonist is (4-(2-(2-aza-bicyclo[2.2.1]hept-2-yl)-ethoxy)-phenyl)-(6-hydroxy-2-(4-hydroxy-phenyl)-benzo[b]thiophen-3-yl)-methanone which is disclosed, along with methods of preparation, in PCT publication no. WO 95/10513 assigned to Pfizer Inc.
Other preferred estrogen agonist/antagonists include compounds as described in commonly assigned U.S. Pat. No. 5,552,412, the disclosure of which is incorporated herein by reference.
Especially preferred compounds described therein are:
cis-6-(4-fluoro-phenyl)-5-(4-(2-piperidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol;
(xe2x88x92)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol(lasofoxifene);
cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol(lasofoxifene);
cis-1-(6xe2x80x2-pyrrolodinoethoxy-3xe2x80x2-pyridyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydronaphthalene;
1-(4xe2x80x2-pyrrolidinoethoxyphenyl)-2-(4xe2x80x3-fluorophenyl)-6-hydroxy-1,2,3,4-tetrahydroisoquinoline;
cis-6-(4-hydroxyphenyl)-5-(4-(2-piperidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro-naphthalene-2-ol; and
1-(4xe2x80x2-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoquinoline.
Other estrogen agonist/antagonists are described in U.S. Pat. No. 4,133,814 (the disclosure of which is incorporated herein by reference). U.S. Pat. No. 4,133,814 discloses derivatives of 2-phenyl-3-aroyl-benzothiophene and 2-phenyl-3-aroylbenzothiophene-1-oxide.
Those skilled in the art will recognize that other bone anabolic agents, also referred to as bone mass augmenting agents, may be used in conjunction with the compounds of this invention. A bone mass augmenting agent is a compound that augments bone mass to a level which is above the bone fracture threshold as detailed in the World Health Organization Study World Health Organization, xe2x80x9cAssessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis (1994). Report of a WHO Study Group. World Health Organization Technical Series 843.xe2x80x9d
Any prostaglandin, or prostaglandin agonist/antagonist may be used as the second compound in certain aspects of this invention. This includes utilizing two different compounds of Formula I of this inventon. Those skilled in the art will recognize that IGF-1, sodium fluoride, parathyroid hormone (PTH), active fragments of parathyroid hormone, growth hormone or growth hormone secretagogues may also be used. The following paragraphs describe exemplary second compounds of this invention in greater detail.
Any prostaglandin-may be used as the second compound in certain aspects of this invention. The term prostaglandin refers to compounds which are analogs of the natural prostaglandins PGD1, PGD2, PGE2, PGE1 and PGF2 which are useful in the treatment of osteoporosis. These compounds bind to the prostaglandin receptors. Such binding is readily determined by those skilled in the art of standard assays (e.g., An S. et al., Cloning and Expression of the EP2 Subtype of Human Receptors for Prostaglandin E2, Biochemical and Biophysical Research Communications, 1993, 197(1):263-270).
Prostaglandins are alicyclic compounds related to the basic compound prostanoic acid. The carbon atoms of the basic prostaglandin are numbered sequentially from the carboxylic carbon atom through the cyclopentyl ring to the terminal carbon atom on the adjacent side chain. Normally the adjacent side chains are in the trans orientation. The presence of an oxo group at C-9 of the cyclopentyl moiety is indicative of a prostaglandin within the E class while PGE2 contains a trans unsaturated double bond at the C13-C14 and a cis double bond at the C5-C6 position.
A variety of prostaglandins are described and referenced below. However, other prostaglandins will be known to those skilled in the art. Exemplary prostaglandins are disclosed in U.S. Pat. Nos. 4,171,331 and 3,927,197, the disclosures of each of which are incorporated herein by reference.
Norrdin et al., The Role of Prostaglandins in Bone In Vivo, Prostaglandins Leukotriene Essential Fatty Acids 41, 139-150, 1990 is a review of bone anabolic prostaglandins.
Any prostaglandin agonist/antagonist may be used as the second compound in certain aspects of this invention. The term prostaglandin agonist/antagonist refers to compounds which bind to prostaglandin receptors (e.g., An S. et al., Cloning and Expression of the EP2 Subtype of Human Receptors for Prostaglandin E2, Biochemical and Biophysical Research Communications, 1993, 197(1):263-270) and mimic the action of prostaglandin in vivo (e.g., stimulate bone formation and increase bone mass). Such actions are readily determined by those skilled in the art of standard assays. Eriksen E. F. et al., Bone Histomorphometry, Raven Press, New York, 1994, pages 1-74; Grier S. J. et. al., The Use of Dual-Energy X-Ray Absorptiometry In Animals, Inv. Radiol., 1996, 31(1):50-62; Wahner H. W. and Fogelman I., The Evaluation of Osteoporosis: Dual Energy X-Ray Absorptiometry in Clinical Practice., Martin Dunitz Ltd., London 1994, pages 1-296. A variety of these compounds are described and referenced below. However, other prostaglandin agonists/antagonists will be known to those skilled in the art. Exemplary prostaglandin agonists/antagonists are disclosed as follows.
Commonly assigned U.S. Pat. No. 3,932,389, the disclosure of which is incorporated herein by reference, discloses 2-descarboxy-2-(tetrazol-5-yl)-11-desoxy-15-substituted-omega-pentanorprostaglandins useful for bone formation activity.
Commonly assigned U.S. Pat. No. 4,018,892, the disclosure of which is incorporated herein by reference, discloses 16-aryl-13,14-dihydro-PGE2 p-biphenyl esters useful for bone formation activity.
Commonly assigned U.S. Pat. No. 4,219,483, the disclosure of which is incorporated herein by reference, discloses 2,3,6-substituted-4-pyrones useful for bone formation activity.
Commonly assigned U.S. Pat. No. 4,132,847, the disclosure of which is incorporated herein by reference, discloses 2,3,6-substituted-4-pyrones useful for bone formation activity.
U.S. Pat. No. 4,000,309, the disclosure of which is incorporated herein by reference, discloses 16-aryl-13,14-dihydro-PGE2 p-biphenyl esters useful for bone formation activity.
U.S. Pat. No. 3,982,016, the disclosure of which is incorporated herein by reference, discloses 16-aryl-13,14-dihydro-PGE2 p-biphenyl esters useful for bone formation activity.
U.S. Pat. No. 4,621,100, the disclosure of which is incorporated herein by reference, discloses substituted cyclopentanes useful for bone formation activity.
U.S. Pat. No. 5,216,183, the disclosure of which is incorporated herein by reference, discloses cyclopentanones useful for bone formation activity.
Sodium fluoride may be used as the second compound in certain aspects of this invention. The term sodium fluoride refers to sodium fluoride in all its forms (e.g., slow release sodium fluoride, sustained release sodium fluoride). Sustained release sodium fluoride is disclosed in U.S. Pat. No. 4,904,478, the disclosure of which is incorporated herein by reference. The activity of sodium fluoride is readily determined by those skilled in the art of biological protocols (e.g., see Eriksen E. F. et al., Bone Histomorphometry, Raven Press, New York, 1994, pages 1-74; Grier S. J. et. al., The Use of Dual-Energy X-Ray Absorptiometry In Animals, Inv. Radiol., 1996, 31(1):50-62; Wahner H. W. and Fogelman I., The Evaluation of Osteoporosis: Dual Energy X-Ray Absorptiometry in Clinical Practice., Martin Dunitz Ltd., London 1994, pages 1-296).
Bone morphogenetic protein may be used as the second compound of this invention (e.g., see Ono, et al., Promotion of the Osteogenetic Activity of Recombinant Human Bone Morphogenetic Protein by Prostaglandin E1, Bone, 1996, 19(6), 581-588).
Any parathyroid hormone (PTH) may be used as the second compound in certain aspects of this invention. The term parathyroid hormone refers to parathyroid hormone, fragments or metabolites thereof and structural analogs thereof which can stimulate bone formation and increase bone mass. Also included are parathyroid hormone related peptides and active fragments and analogs of parathyroid related peptides (see PCT publication no. WO 94/01460). Such bone anabolic functional activity is readily determined by those skilled in the art of standard assays (e.g., see Eriksen E. F. et al., Bone Histomorphometry, Raven Press, New York, 1994, pages 1-74; Grier S. J. et. al., The Use of Dual-Energy X-Ray Absorptiometry In Animals, Inv. Radiol., 1996, 31(1):50-62; Wahner H. W. and Fogelman I., The Evaluation of Osteoporosis: Dual Energy X-Ray Absorptiometry in Clinical Practice., Martin Dunitz Ltd., London 1994, pages 1-296). A variety of these compounds are described and referenced below. However, other parathyroid hormones will be known to those skilled in the art. Exemplary parathyroid hormones are disclosed in the following references.
xe2x80x9cHuman Parathyroid Peptide Treatment of Vertebral Osteoporosisxe2x80x9d, Osteoporosis lnt., 3, (Supp 1):199-203.
xe2x80x9cPTH 1-34 Treatment of Osteoporosis with Added Hormone Replacement Therapy: Biochemical, Kinetic and Histological Responsesxe2x80x9d Osteoporosis Int. 1:162-170.
Any growth hormone or growth hormone secretagogue may be used as the second compound in certain aspects of this invention. The term growth hormone secretagogue refers to a compound which stimulates the release of growth hormone or mimics the action of growth hormone (e.g., increases bone formation leading to increased bone mass). Such actions are readily determined by those skilled in the art of standard assays well known to those of skill in the art. A variety of these compounds are disclosed in the following published PCT patent applications: WO 95/14666; WO 95/13069; WO 94/19367; WO 94/13696; and WO 95/34311. However, other growth hormones or growth hormone secretagogues will be known to those skilled in the art.
In particular a preferred growth hormone secretagogue is N-[1(R)-[1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4xe2x80x2-piperidin]-1xe2x80x2-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide:MK-677.
Other preferred growth hormone secretagogues include
2-amino-N-(2-(3a-(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo-[4,3-c]pyridin-5-yl)-1-(R)-benzyloxymethyl-2-oxo-ethyl)-isobutyramide or its L-tartaric acid salt;
2-amino-N-(1-(R)-benzyloxymethyl-2-(3a-(R)-(4-fluoro-benzyl)-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-2-oxo-ethyl)isobutyramide;
2-amino-N-(2-(3a-(R)-benzyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-1-(R)benzyloxymethyl-2-oxo-ethyl)isobutyramide; and
2-amino-N-(1-(2,4-difluoro-benzyloxymethyl)-2-oxo-2-(3-oxo-3a-pyridin-2-ylmethyl-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-ethyl)-2-methyl-propionamide.
The term xe2x80x9cHMG-CoA reductase inhibitorxe2x80x9d is intended to include compounds which inhibit the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Any HMG-CoA reductase inhibitor may be used as the second compound of this invention, including mevastatin, lovastatin, pravastatin, velostatin, simvastatin, fluvastatin, cerivastatin, mevastatin, dalvastatin, fluindostatin and atorvastatin, or a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug.
Statins enhance the production of osteoblasts, the cells that produce new bone. The expression of the bone growth factor Bone Morphogenetic Protein (BMP) is known to enhance osteoblast differentiation. S. E. Harris, et al., Mol. Cell. Differ. 3, 137 (1995). Statins are in turn found to enhance BMP production. G. Mundy, et al., Stimulation of Bone Formation in Vitro and in Rodents by Statins, Science, 286, 1946 (1999). Mundy, et al. find that statins increase new bone formation as well as increase osteoblast cell numbers at all stages of differentiation.
It is preferred that said statin is mevastatin, lovastatin, pravastatin, velostatin, simvastatin, fluvastatin, cerivastatin, mevastatin, dalvastatin, fluindostatin or atorvastatin, or a prodrug thereof, or a pharmaceutically acceptable salt of said compound or prodrug.
It is especially preferred that said statin is atorvastatin, most preferably atorvastatin calcium.
HMG-CoA reductase inhibitors may be readily prepared by processes known in the chemical arts. Mevastatin, lovastatin, pravastatin, velostatin, simvastatin, fluvastatin, cerivastatin and mevastatin, dalvastatin and fluindostatin may be made in accordance with the process set forth in U.S. Pat. Nos. 3,983,140, 4,231,938, 4,346,227, 4,448,784, 4,450,171, 4,739,073, 5,177,080, 5,177,080, European Patent Application No. 738,510 A2 and European Patent Application No. 363,934 A1 respectively, which are all incorporated herein by reference.
Atorvastatin may readily be prepared as described in U.S. Pat. No. 4,681,893, which is incorporated herein by reference. The hemicalcium salt of atorvastatin, which is currently sold as Lipitor(copyright), may readily be prepared as described in U.S. Pat. No. 5,273,995, which is incorporated herein by reference. Other pharmaceutically-acceptable cationic salts of atorvastatin may be readily prepared by reacting the free acid form of atorvastatin with an appropriate base, usually one equivalent, in a co-solvent.
Administration of the EP4 receptor selective agonists according to the methods of this invention can be via any mode which delivers the EP4 receptor selective agonist systemically and/or locally (e.g., at the site of the bone fracture, osteotomy, or orthopedic surgery). These methods include oral routes, parenteral, intraduodenal routes, etc. Generally, the compounds of this invention are administered orally, but parenteral administration (e.g., intravenous, intramuscular, transdermal, subcutaneous, rectal or intramedullary) may be utilized, for example, where oral administration is inappropriate for the target or where the patient is unable to ingest the drug.
The methods of this invention are used for the treatment and promotion of healing of bone fractures and osteotomies by the local application (e.g., to the sites of bone fractures of osteotomies) of EP4 receptor selective agonists. The EP4 receptor selective agonists of this invention are applied to the sites of bone fractures or osteotomies, for example, either by injection of the compound in a suitable solvent (e.g., an oily solvent such as arachis oil) to the cartilage growth plate or, in cases of open surgery, by local application thereto of the compound in a suitable vehicle, carrier or diluent such as bone-wax, demineralized bone powder, polymeric bone cements, bone sealants, etc. Alternatively, local application can be achieved by applying a solution or dispersion of the compound in a suitable carrier or diluent onto the surface of, or incorporating it into solid or semi-solid implants conventionally used in orthopedic surgery, such as dacron-mesh, gel-foam and kiel bone, or prostheses.
In any event, the amount and timing of compounds administered will, of course, be dependent on the subject being treated, on the severity of the affliction, on the manner of administration and on the judgment of the prescribing physician. Thus, because of patient to patient variability, the dosages given herein are a guideline and the physician may titrate doses of the compound to achieve the treatment (e.g., bone mass augmentation) that the physician considers appropriate for the patient. In considering the degree of treatment desired, the physician must balance a-variety of factors such as bone mass starting level, age of the patient, presence of preexisting disease, as well as presence of other diseases (e.g., cardiovascular disease).
In general, an amount of a compound of Formula I of this invention is used that is sufficient to augment bone mass to a level which is above the bone fracture threshold (as detailed in the World Health Organization Study previously cited herein).
The compounds used in the methods of this invention are generally administered in the form of a pharmaceutical composition comprising at least one of the compounds of this invention together with a pharmaceutically acceptable carrier, vehicle or diluent. Thus, the EP4 receptor selective agonist can be administered individually in any conventional local, oral, intranasal, parenteral, rectal or transdermal dosage form.
For oral administration the pharmaceutical composition can take the form of solutions, suspensions, tablets, pills, capsules, powders, and the like. Tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate are employed along with various disintegrants such as starch and preferably potato or tapioca starch and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as, magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type are also employed as fillers in soft and hard-filled gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the compositions of this invention can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents and/or suspending agents, as well as such diluents as water, ethanol, propylene glycol, glycerin or various like combinations thereof.
For purposes of parenteral administration, solutions in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.
For purposes of transdermal (e.g.,topical) administration, dilute sterile, aqueous or partially aqueous solutions (usually in about 0.1% to 5% concentration), otherwise similar to the above parenteral solutions, are prepared.
Methods of preparing various pharmaceutical compositions with a certain amount of active ingredient are known, or will be apparent in light of this disclosure, to those skilled in the art. For examples of methods of preparing pharmaceutical compositions, see Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th Edition (1995).
NMR spectra were recorded on a Varian Unity 400 spectrometer (Varian Co., Palo Alto, Calif.) at about 23xc2x0 C. at 400 MHz for proton nuclei. Chemical shifts are expressed in parts per million. The peak shapes are denoted as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; bs, broad singlet. Atmospheric pressure chemical ionization (APCl) mass spectra were obtained on a Fisons Platform II Spectrometer (Micromass Inc., Beverly, Mass.). Where the intensity of chlorine or bromine-containing ions are described the expected intensity ratio was observed (approximately 3:1 for 35Cl/37Cl-containing ions) and 1:1 for 79Br/81Br-containing ions) and the intensity of only the lower mass ion is given.
Medium pressure chromatography was performed using a Biotage purification system (Biotage, Dyax Corporation, Charlottesville, Va.) under nitrogen pressure. Flash chromatography was performed with either Baker Silica Gel (40 xcexcm) (J. T. Baker, Phillipsburg, N.J.) or Silica Gel 60 (EM Sciences, Gibbstown, N.J.) in glass columns under low nitrogen pressure. Radial Chromatography was performed using a Chromatotron (model 7924T, Harrison Research, Palo Alto, Calif.). Preparative Chromatography was performed using Analtech Uniplates Silica Gel GF (20xc3x9720 cm) (Analtech, Inc. Newark, Del.). Dimethylformamide (DMF), tetrahydrofuran (THF), and dichloromethane (CH2Cl2) used as reaction solvents were the anhydrous grade supplied by Aldrich Chemical Company (Milwaukee, Wis.). The term xe2x80x9cconcentratedxe2x80x9d refers to removal of solvent at water aspirator pressure on a rotary evaporator. The term xe2x80x9cEtOAcxe2x80x9d means ethyl acetate. The abbreviation xe2x80x98hxe2x80x99 stands for hours. The term xe2x80x9cTBAFxe2x80x9d refers to tetrabutylammonium fluoride. The term xe2x80x9cDMAPxe2x80x9d refers to dimethylaminopyridine. The terms xe2x80x9cdichloromethanexe2x80x9d and xe2x80x9cmethylene chloridexe2x80x9d are synonymous and are used interchangeably throughout this description and in the Examples and Preparations.
NMR spectra were recorded on a Varian Unity 400 spectrometer (Varian Co., Palo Alto, Calif.) at about 23xc2x0 C. at 400 MHz for proton nuclei. Chemical shifts are expressed in parts per million. The peak shapes are denoted as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; bs, broad singlet. Atmospheric pressure chemical ionization (APCl) mass spectra were obtained on a Fisons Platform II Spectrometer (Micromass Inc., Beverly, Mass.). Where the intensity of chlorine or bromine-containing ions are described the expected intensity ratio was observed (approximately 3:1 for 35Cl/37Cl-containing ions) and 1:1 for 79Br/81Br-containing ions) and the intensity of only the lower mass ion is given.
Medium pressure chromatography was performed using a Biotage purification system (Biotage, Dyax Corporation, Charlottesville, Va.) under nitrogen pressure. Flash chromatography was performed with either Baker Silica Gel (40 xcexcm) (J. T. Baker, Phillipsburg, N.J.) or Silica Gel 60 (EM Sciences, Gibbstown, N.J.) in glass columns under low nitrogen pressure. Radial Chromatography was performed using a Chromatotron (model 7924T, Harrison Research, Palo Alto, Calif.). Preparative Chromatography was performed using Analtech Uniplates Silica Gel GF (20xc3x9720 cm) (Analtech, Inc. Newark, Del.). Dimethylformamide (DMF), tetrahydrofuran (THF), and dichloromethane (CH2Cl2) used as reaction solvents were the anhydrous grade supplied by Aldrich Chemical Company (Milwaukee, Wis.). The term xe2x80x9cconcentratedxe2x80x9d refers to removal of solvent at water aspirator pressure on a rotary evaporator. The term xe2x80x9cEtOAcxe2x80x9d means ethyl acetate. The abbreviation xe2x80x98hxe2x80x99 stands for hours. The term xe2x80x9cTBAFxe2x80x9d refers to tetrabutylammonium fluoride. The term xe2x80x9cDMAPxe2x80x9d refers to dimethylaminopyridine. The terms xe2x80x9cdichloromethanexe2x80x9d and xe2x80x9cmethylene chloridexe2x80x9d are synonymous and are used interchangeably throughout this description and in the Examples and Preparations.